Kinase inhibitors and uses thereof

ABSTRACT

The present invention relates to kinase inhibiting compositions and uses thereof. The invention further provides isolated kinase inhibiting peptides and uses thereof for inhibiting hyperplasia, for inhibiting the growth of neoplasms, and for inducing programmed cell death in a cell population.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Applications60/963,941, filed Aug. 7, 2007, and 60/994,970, filed Sep. 24, 2007,each of which is incorporated by reference in its entirety herein.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with government support under NIH/NHLBI GrantNumber HLO74968 awarded by the National Institute of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to cell biology, uses of kinase inhibitingpeptides and nucleic acids which encode peptides, and therapeuticmethods of use thereof.

BACKGROUND

Kinases

Kinases are a ubiquitous group of enzymes that catalyze the phosphorylgroup transfer reaction from a phosphate donor (usually ATP) to areceptor substrate. Although all kinases catalyze essentially the samephosphoryl transfer reaction, they display remarkable diversity in theirsubstrate specificity, structure, and the pathways in which theyparticipate. A recent classification of all available kinase sequences(approximately 60,000 sequences) indicates that kinases may be groupedinto 25 families of homologous proteins. These kinase families areassembled into 12 fold groups based on similarity of structural fold. 22of the 25 families (approximately 98.8% of all sequences) belong to 10fold groups for which the structural fold is known. Of the other 3families, polyphosphate kinase forms a distinct fold group; the 2remaining families are both integral membrane kinases and comprise thefinal fold group. These fold groups not only include some of the mostwidely spread protein folds, such as Rossmann-like fold, ferredoxin-likefold, TIM-barrel fold, and antiparallel β-barrel fold, but also allmajor classes (all α, all β, α+β, α/β) of protein structures. Within afold group, the core of the nucleotide-binding domain of each family hasthe same architecture, and the topology of the protein core is eitheridentical or related by circular permutation. Homology between thefamilies within a fold group is not implied.

Group I (23,124 sequences) kinases incorporate protein S/T-Y kinase,atypical protein kinase, lipid kinase, and ATP grasp enzymes and furthercomprise the protein S/T-Y kinase, and atypical protein kinase family(22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32);protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38);homoserine kinase (EC 2.7.1.39); 1-phosphatidylinositol 4-kinase (EC2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH₂)] kinase (EC2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitratedehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain]kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119);calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsinkinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126);[myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); 1-phosphatidylinositol3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141);phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); andphosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group Ifurther comprises the lipid kinase family (321 sequences). These kinasesinclude: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); ID-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127);inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140);1-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149);1-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150);inositol-polyphosphate multikinase (EC 2.7.1.151); andinositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprisesthe ATP-grasp kinases (729 sequences), which includeinositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphatedikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).

Group II (17,071 sequences) kinases incorporate the Rossman-likekinases. Group II comprises: (i) the P-loop kinase family (7,732sequences), which include gluconokinase (EC 2.7.1.12);phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21);ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate kinase (EC2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC2.7.1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase (EC2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); polynucleotide5′-hydroxyl-kinase (EC 2.7.1.78); 6-phosphofructo-2-kinase (EC2.7.1.105); deoxyguanosine kinase (EC 2.7.1.113); tetraacyldisaccharide4′-kinase (EC 2.7.1.130); deoxynucleoside kinase (EC 2.7.1.145);adenosylcobinamide kinase (EC 2.7.1.156); polyphosphate kinase (EC2.7.4.1); phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate kinase (EC2.7.4.8); thymidylate kinase (EC 2.7.4.9);nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10);(deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC2.7.4.14); and uridylate kinase (EC 2.7.4.-); (ii) thephosphoenolpyruvate carboxykinase family (815 sequences), which includesprotein kinase (HPr kinase/phosphatase) (EC 2.7.1.37);phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); andphosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49); (iii) thephosphoglycerate kinase (1,351 sequences) family, which includesphosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate kinase (GTP)(EC 2.7.2.10); (iv) the aspartokinase family (2,171 sequences), whichincludes carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4);acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1)and uridylate kinase (EC 2.7.4.-); (v) the phosphofructokinase-likekinase family (1,998 sequences), which includes 6-phosphofrutokinase (EC2.7.1.1 1); NAD(+) kinase (EC 2.7.1.23); 1-phosphofructokinase (EC2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase (EC2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC2.7.1.107); and ceramide kinase (EC 2.7.1.138); (vi) the ribokinase-likefamily (2,722 sequences), which includes glucokinase (EC 2.7.1.2);ketohexokinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4);6-phosphofructokinase (EC 2.7.1. 11); ribokinase (EC 2.7.1.15);adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35);2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidinekinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50);1-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73);5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphatekinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146);ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidinekinase (EC 2.7.4.7); (vii) the thiamin pyrophosphokinase family (175sequences), which includes thiamin pyrophosphokinase (EC 2.7.6.2); and(viii) the glycerate kinase family (107 sequences), which includesglycerate kinase (EC 2.7.1.31).

Group III kinases (10,973 sequences) comprise (i) the ferredoxin-likefold kinases; (ii) the nucleoside-diphosphate kinase family (923sequences), which includes nucleoside-diphosphate kinase (EC 2.7.4.6);(iii) the HPPK kinase family (609 sequences), which includes2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC2.7.6.3); (iv) the guanido kinase family (324 sequences), which includesguanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2);arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5); (v)the histidine kinase family (9,117 sequences), which includes proteinkinase (histidine kinase) (EC 2.7.1.37); [pyruvatedehydrogenase(lipoamide)] kinase (EC 2.7.1.99); and[3-methyl-2-oxybutanoate dehydrogenase(lipoamide)] kinase (EC2.7.1.115).

Group IV kinases (2,768 sequences) incorporate H-like kinases, whichinclude hexokinase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase(EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase (EC 2.7.1.7);gluconokinase (EC 2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase(EC 2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53);allose kinase (EC 2.7.1.55); 2-dehydro-3-deoxygalactonokinase (EC2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1.59); N-acylmannosaminekinase (EC 2.7.1.60); polyphosphate-glucose phosphotransferase (EC2.7.1.63); beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC2.7.2.1); butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase(EC 2.7.2.14); and propionate kinase (EC 2.7.2.-).

Group V kinases (1,119 sequences) incorporate TIM β/α-barrel kinases,which include pyruvate kinase (EC 2.7.1.40).

Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymesinclude galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36);homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46);fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); andphosphomevalonate kinase (EC 2.7.4.2)

Group VII kinases (1,843 sequences) incorporate AIR synthetase-likekinases, which include thiamine-phosphate kinase (EC 2.7.4.16) andselenide, water dikinase (EC 2.7.9.3).

Group VIII kinases (565 sequences) incorporate riboflavin kinases (565sequences), which include riboflavin kinase (EC 2.7.1.26).

Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases,which include glycerone kinase (EC 2.7.1.29).

Group X kinases (148 sequences) incorporate putative glycerate kinases,which include glycerate kinase (EC 2.7.1.31).

Group XI kinases (446 sequences) incorporate polyphosphate kinases,which include polyphosphate kinases (EC 2.7.4.1).

Group XII kinases (263 sequences) incorporate integral membrane kinases.Group XII comprises the dolichol kinase family, which include dolicholkinases (EC 2.7.1.108); and the undecaprenol kinase family, whichinclude undecaprenol kinases (EC 2.7.1.66).

Kinases, which are among the best-studied enzymes at the structural,biochemical, and cellular levels, play indispensable roles in numerouscellular metabolic and signaling pathways. Even though all kinases usethe same phosphate donor (in most cases, ATP) and appear to catalyzeapparently the same phosphoryl transfer reaction, they displayremarkable diversity in their structural folds and substrate recognitionmechanisms. This is probably due largely to the extraordinarily diversenature of the structures and properties of their substrates.

Signal Transduction Pathways

The AGC family of protein kinases, which comprise isoforms of proteinkinase B (PKB, also known as Akt), p70 ribosomal S6 kinase (S6K), serum-and glucocorticoid-induced protein kinase (SGK), and atypical isoformsof protein kinase C (PKC), are activated within minutes of insulin- orgrowth factor induced stimulation of phosphatidylinositol 3-kinase(PI₃-kinase). Once activated, PKB/Akt phosphorylates and modulates thefunction of a number of important regulatory proteins, resulting ininhibition of apoptosis, formation of cell division and stimulation ofglucose uptake and storage. The serine/threonine kinase Akt (proteinkinase B) is a critical enzyme in signal transduction pathways involvedin cell proliferation, apoptosis, angiogenesis, and diabetes. Threeisoforms of Akt (α, β, γ or Akt 1, 2, 3) are known in mammals. Theseisoforms exhibit a high degree of homology but differ slightly in thelocalization of their regulatory phosphorylation sites. The Akt enzymesare composed of three functionally distinct regions: 1) an N-terminalpleckstrin homology (PH) domain; 2) a central catalytic domain; and 3) aC-terminal hydrophobic motif. The PH domain in the N-terminal region ofAkt provides a lipid binding module to direct Akt to PIP₂ (phosphatidylinositol bisphosphate or the products obtained by cleavage of PIP₃) andPIP₃ (phosphatidyl inositol (3,4,5)-triphosphate, the product of theclass I phosphoinositide 3-kinase activity on phosphatidyl inositol(4,5)-bisphosphate), interacts with 3′-phosphoinositides and helps torecruit Akt to the plasma membrane.

In unstimulated cells, Akt is constitutively phosphorylated at Ser¹²⁴,in the region between the PH and catalytic domains, and on Thr⁴⁵⁰, inthe C-terminal region (in Aktα). Activation of Akt involves growthfactor binding to a receptor tyrosine kinase and activation of PI 3-K(PI 3-K phosphorylates membrane bound PIP₂ to generate PIP₃). Thebinding of PIP₃ to the PH domain anchors Akt to the plasma membrane andallows its phosphorylation and activation by PDK1 (pyruvatedehydrogenase kinase isozyme 1). Akt is fully activated following itsphosphorylation at two regulatory residues, a threonine residue on thekinase domain and a serine residue on the hydrophobic motif. Thesemotifs are structurally and functionally conserved within the AGC kinasefamily. Phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ is required for theactivation of Aktα. Phosphorylation at Thr³⁰⁹ and Ser⁴⁷⁴ activates Aktβ.Phosphorylation at Thr³⁰⁵ activates Aktγ. Akt activation requiresphosphorylation of a threonine residue on the kinase domain, catalyzedby PDK1. This causes a charge-induced conformational change, and allowssubstrate binding and an increased rate of catalysis. Phosphorylation atthe serine residue, primarily by mTOR/richtor complex (mTORC₂), augmentsAkt activity by approximately 10-fold. Studies indicate that DNA-PK andPKCbII phosphorylate the serine residue on the regulatory subunit. Thehydrophobic motif of Akt, without threonine phosphorylation, is moresusceptible to the action of phosphatases; however, the duallyphosphorylated and fully active enzyme is stable, allowing itslocalization to the nucleus and other sites. The activity of Akt isnegatively regulated by PTEN (phosphatase and tensin homolog gene whoseproduct acts as a phosphatase to dephosphorylate phosphatidylinositol(3,4,5)-triphosphate) and SHIP(SH2-containing inositol phosphatase,INPP5D).

Akt facilitates growth factor-mediated cell survival and blocksapoptotic cell death by deactivating (via phosphorylation) pro-apoptoticfactors such as Bad, caspase-9, and Forkhead transcription factors (AFX,Daf-16, FKHR). The phosphorylation of Bad at Ser¹³⁶ promotes itsassociation with 14-3-3 proteins in the cytosol; this prevents Bad fromlocalizing at the mitochondria to induce apoptosis. Akt promotes cellsurvival by inactivating caspase-9 via phosphorylation at Ser¹⁹⁶.Similarly, activated Akt phosphorylates Forkhead family members,resulting in their sequestration in the cytoplasm. In the absence ofsurvival factors and Akt activity, Forkhead family members translocateto the nucleus, wherein they initiate a program of gene expression (forexample, FasL) that promotes cell death. Akt also phosphorylates andactivates IKKα (a subunit of 1κB alpha kinase complex that has animportant role in the activation of nuclear factor-κB (NF-κB), a keyregulator of normal and tumor cell proliferation, apoptosis and responseto chemotherapy) at Thr²³. The activated IKKα, in turn, phosphorylatesIκB, targeting it for ubiquitination and proteasomal degradation. Thisleads to the activation and nuclear translocation of NF-κB, and thetranscription of NF-kB-dependent pro-survival genes which includeBcl-x_(L) and caspase inhibitors.

Akt also phosphorylates and inactivates GSK-3 (glycogen synthasekinase-3), allowing the activation of glycogen synthase to proceed.GSK-3 phosphorylates cyclin D (a regulator of G1 to S phase transition),targeting cyclin D for proteolysis. Thus, the inactivation of GSK-3 maypromote the up-regulation of cyclin D and enhance cell cycling.

Studies indicate Akt phosphorylates Chk1 (a DNA damage effector kinase)at Ser²⁸⁰ thereby preventing human protein kinases ATM (ataxiaAtaxiatelangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3 related)from activating Chk1 via phosphorylation at Ser³⁴⁵. This may be oftherapeutic significance as Chk1 inhibition enhances sensitization oftumors to chemotherapeutic agents.

Akt phosphorylates Cdc25B on Ser³⁵³, resulting in its cytoplasmicaccumulation. Cdc25B undergoes activation during S-phase and has a rolein activating the mitotic kinase Cdk1/cyclin B in the cytoplasm. Thisrelocation of Cdc25B to the cytoplasm allows Akt to regulate Cdc25Bfunction and participate in controlling the entry of cells into mitosis.

The regulation of Akt by a number of upstream oncogenes and tumorsuppressor genes influences cancer progression. Breast cancer cell linesexpress Aktα in varying degrees. The Akt inhibitor,1L-6-hydroxymethylchiro-inositol2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduces survival of both drugresistant and drug sensitive multiple myeloma cells. Akt also has acritical role in tumorigenesis. Akt is activated when tumor suppressors,such as cell cycle inhibitor p27 and PTEN, lose their functions. Aktimpairs the nuclear import of p27 by phosphorylation of p27 at Thr¹⁵⁷.Cytoplasmic mislocalization of p27 has been strongly linked to loss ofdifferentiation and poor outcome in breast cancer. Akt also is reportedto associate physically with endogenous p21 (a cell cycle inhibitor).The phosphorylation of p21 at Thr¹⁴⁵ by Akt causes p21 localization tothe cytoplasm and subsequent degradation.

Akt and p53 (also known as protein 53 or tumor protein 53, atranscription factor that regulates the cell cycle) have opposing rolesin signaling pathways that determine cell survival. Under conditionswhere the apoptotic effect of p53 is dominant, destruction of Akt has arole in accelerating the apoptotic process. In apoptosis-prone cells,p53-dependent signaling enables downregulation of Akt, which predisposescells to rapid apoptosis in response to stress signals. Under certaincircumstances, Akt activation may overcome the death promoting effectsof p53 and may rescue cells from apoptosis. Studies indicate that Aktcan phosphorylate Mdm2 (a protein encoded by an oncogene that modulatesp53 tumor suppressor activity) on Ser¹⁶⁶ and Ser¹⁸⁸ and promote Mdm2translocation to the nucleus wherein Mdm2 destabilizes p53 and enhancesits degradation via the proteasomal pathway.

Kinase Inhibition

The eukaryotic protein kinases constitute one of the largestsuperfamilies of homologous proteins that are related to each other byvirtue of their catalytic domains. Most related protein kinases arespecific for either serine/threonine phosphorylation or tyrosinephosphorylation. Stimulation of protein kinases is considered to be oneof these enzymes most common activation mechanisms in signaltransduction systems and therefore plays an integral role in thecellular response to extracellular stimuli. Many substrates are known toundergo phosphorylation by multiple protein kinases. A considerableamount of information on primary sequence of the catalytic domains ofvarious protein kinases has been published. These sequences share alarge number of residues involved in ATP binding, catalysis, andmaintenance of structural integrity. Most protein kinases possess a wellconserved 30-32 kDa catalytic domain. Studies have attempted to identifyand utilize regulatory elements of protein kinases. These regulatoryelements include antibodies, blocking peptides, and inhibitors.

Inhibitors

Enzyme inhibitors are molecules that bind to enzymes thereby decreasingenzyme activity. The binding of an inhibitor may stop substrate fromentering the active site of the enzyme and/or hinder the enzyme fromcatalyzing its reaction. Inhibitor binding is either reversible orirreversible. Irreversible inhibitors usually react with the enzyme andchange it chemically, for example, by modifying key amino acid residuesneeded for enzymatic activity. In contrast, reversible inhibitors bindnon-covalently and produce different types of inhibition depending onwhether these inhibitors bind the enzyme, the enzyme-substrate complex,or both.

Enzyme inhibitors often are evaluated by their specificity and potency.The term “specificity” as used herein refers to the selective attachmentor influence of one substance on another. The term “potency” as usedherein refers to efficacy, effectiveness, strength, or, typically, thedissociation constant, which indicates the concentration needed toinhibit an enzyme.

Inhibitors of protein kinases have been studied for use as tools inprotein kinase activity regulation Inhibitors have been studied for usewith, for example, cyclin-dependent (Cdk) kinase, MAP kinase,serine/threonine kinase, Src Family protein tyrosine kinase, tyrosinekinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase(Chk1), GSK-3, INK, MEK, myosin light chain kinase (MLCK), proteinkinase A, Akt (protein kinase B), protein kinase C, protein kinase G,protein tyrosine kinase, Raf kinase, and Rho kinase.

Antibodies

Antibodies (or “immunoglobulins”) are gamma globulin proteins producedby B lymphocytes of the immune system in response to an antigen used bythe body to identify and neutralize foreign objects having that antigen.In their native form, they are typically made of basic structuralunits—each with two large heavy (H) chains and two small light (L)chains—to form, for example, monomers with one unit, dimers with twounits or pentamers with five units. There are several different types ofantibody heavy chains, and several different kinds of antibodies, whichare grouped into different isotypes based on which heavy chain theypossess. Five different antibody isotypes are known in mammals, whichperform different roles, and help direct the appropriate immune responsefor each different type of foreign object they encounter. Thespecificity and binding affinity of an antibody are dictated by thethree polyvariable loops of the VL chain and the three hypervariableloops of the VH chain located on each arm of that antibody. Variationsin the lengths and sequences of these loops define theantibody-combining site (ACS). As used herein, the term “antibody”includes, by way of example, both naturally occurring and non-naturallyoccurring antibodies. Specifically, the term “antibody” includespolyclonal antibodies and monoclonal antibodies, and fragments thereof.Furthermore, the term “antibody” includes chimeric antibodies and whollysynthetic antibodies, and fragments thereof. The terms “epitope” and“antigenic determinant” are used interchangeably herein to refer to thesite on a molecule that an ACS recognizes and to which that antibodybinds/attaches itself. An epitope may be an antigenicdeterminant/antigen binding site on a kinase inhibiting peptide. Theepitope may be primary, secondary, or tertiary-sequence related.

The specificity of the interactions between certain antibodies andprotein kinases has been studied for use in protein kinase activityregulation. Antibodies have been isolated for use with, for example, MAPkinase pathways, protein kinase A, protein kinase B, protein kinase G,serine/threonine kinases, glycogen-synthase kinase-3 (GSK-3),stress-activated protein (SAP) kinase pathways, and tyrosine kinases.Additionally, antibodies have been isolated for use with protein kinaseinhibitors and protein kinase substrates.

Blocking Peptides

A peptide is a chemical compound that is composed of a chain of two ormore amino acids; the carboxyl group of one amino acid is linked to theamino group of an adjacent amino acid to form a peptide bond. The term“polypeptide” is used herein in its broadest sense to refer to asequence of subunit amino acids, amino acid analogs or peptidomimetics,wherein the subunits are linked by peptide bonds. The peptides orpolypeptides may by chemically synthesized or expressed recombinantly.Peptides have been used in the study of protein structure and function.Synthetic peptides may be used as probes to see where protein-peptideinteractions occur. Inhibitory peptides may be used in clinical researchto examine the effects of peptides on the inhibition of protein kinases,cancer proteins and other disorders.

The use of several blocking peptides has been studied. For example,extracellular signal-regulated kinase (ERK), a MAPK protein kinase(meaning any of a group of protein serine/threonine kinases that respondto extracellular stimuli (antigens) and regulate various cellularactivities), is essential for cellular proliferation anddifferentiation. The activation of MAPKs requires a cascade mechanismwhereby MAPK is phosphorylated by an upstream MAPKK (MEK) which is then,in turn phosphorylated by a third kinase MAPKKK (MEKK). This inhibitorypeptide functions as a MEK decoy by binding to ERK. It contains theamino-terminal 13 amino acids (GMPKKKPTPIQLN) [SEQ ID NO: 149] of MEK1and binds to ERK. This blocks ERK activation by MEK as ERK is unable tointeract with MEK. The ERK inhibitory peptide also contains a proteintransduction (PTD) sequence (DRQIKIWFQNRRMKWKK) [SEQ ID NO: 150] derivedfrom Antennapedia which renders the peptide cell permeable.

Other blocking peptides include autocamtide-2 related inhibitory peptide(AIP). This synthetic peptide is a highly specific and potent inhibitorof Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). AIP is anon-phosphorylatable analog of autocamtide-2, a highly selective peptidesubstrate for CaMKII. AIP inhibits CaMKII with an IC₅₀ of 100 nM (IC₅₀is a concentration of the inhibitor required to obtain 50% inhibition).The AIP inhibition is non-competitive with respect to syntide-2 (CaMKIIPeptide Substrate) and ATP but competitive with respect toautocamtide-2. The inhibition is unaffected by the presence or absenceof Ca2+/calmodulin. CaMKII activity is completely inhibited by 1 μM AIP;PKA, PKC and CaMKIV are not affected. The amino acid sequence of AIP is:KKALRRQEAVDAL (Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu) [SEQID NO: 151].

Other blocking peptides include cyclin-dependent kinase 5 (Cdk5)inhibitory peptide (CIP). Cdk5 phosphorylates tau at Alzheimer's Disease(AD)-specific phospho-epitopes when it associates with the p25regulatory component. p25 is a truncated activator of the Cdk-p25heterodimer (a microtubule associated protein), which is produced fromthe physiological Cdk5 activator p35 upon exposure to amyloid-beta (Aβ,a protein implicated in AD) peptides. Upon neuronal infections with CIP,CIPs selectively inhibit p25/Cdk5 activity and suppress the aberrant tauphosphorylation in cortical neurons. The reasons for the specificitydemonstrated by CIP are not fully understood.

Additional blocking peptides have been studied for ERK2, ERK3, p38/HOG1,protein kinase C, casein kinase II, Ca²⁺/calmodulin kinase IV, caseinkinase II, Cdk4, Cdk5, DNA-PK, PAK3, PI-3 kinase, PI-5 kinase, PSTAIRE,ribosomal S6 kinase, GSK-4, GCK, SAPK, SEK1, and FAK.

Protein Transduction Domains

New drug delivery technologies occupy an important niche in treatmentsas they enable drugs to be more effective. Drug delivery still isconsidered a poor relation to drug discovery, with greater than 95% ofall new potential therapeutics having poor pharmacokinetics. Thegreatest impediment for cytosolic release of therapeutic molecules isthe membrane barrier of target cells. Protein transduction domains(PTDs), also referred to as Trojan peptides, membrane translocatingsequences or cell permeable proteins, are a class of peptides that aregenerally capable of penetrating the plasma membrane of mammalian cells.PTDs are generally 10-16 amino acids in length and may be groupedaccording to their composition, for example peptides rich in arginineand/or lysine. PTDs also may be used to assist novel HSP27 kinaseinhibitors to penetrate cell membranes (see, e.g., PCT/US2007/16246,filed Jul. 16, 2007, entitled “Polypeptic Inhibitors of HSP27 and UsesThereof,” which is incorporated by reference herein in its entirety).PTDs are capable of transporting compounds of many types and molecularweights across mammalian cells. These compounds include effectormolecules, such as proteins, DNA, conjugated peptides, oligonucleotides,and small particles such as liposomes. When PTDs are chemically linkedor fused to other proteins these fusion proteins are still able topenetrate the plasma membrane and enter cells. Although the exactmechanism of this transduction is unknown, internalization of theseproteins is not believed to be receptor-mediated ortransporter-mediated. The use of PTDs capable of transporting effectormolecules into cells has become increasingly attractive in the design ofdrugs as they promote the cellular uptake of cargo molecules. These cellpenetrating peptides, generally categorized as amphipathic or cationicdepending on their sequence, provide a non-invasive delivery technologyfor macromolecules.

Viral PTD Containing Proteins

The first proteins to be described as having transduction propertieswere viral in origin. These proteins still are the most commonlyaccepted models for PTD action. The HIV-1 Transactivator oftranscription (TAT) and HSV-1 VP 22 protein are the best characterizedviral PTD containing proteins.

TAT (HIV-1 trans-activator gene product) is an 86-amino acid polypeptidethat act as a powerful transcription factor of the integrated HIV-1genome. TAT stimulates viral replication in latently infected cells. Thetranslocation properties of the TAT protein enable it to activatequiescent infected cells and may be involved in priming of uninfectedcells for subsequent infection by regulating many cellular genes,including cytokines. The minimal PTD of TAT is the 9 amino acid proteinsequence RKKRRQRRR (TAT₄₉₋₅₇) [SEQ ID NO: 152]. Studies utilizing alonger fragment of TAT demonstrated successful transduction of fusionproteins up to 120 kDa. The addition of multiple TAT-PTDs and syntheticTAT derivatives have been demonstrated to mediate membranetranslocation. TAT PTD containing fusion proteins have been used astherapeutic moieties in experiments involving cancer, for transporting adeath-protein into cells, and in disease models of neurodegenerativedisorders.

VP22 is the HSV-1 tegument protein, a structural part of the HSV virion.VP22 is capable of receptor independent translocation and accumulates inthe nucleus. This property of VP22 classifies the protein as a PTDcontaining peptide. Fusion proteins comprising full length VP22 havebeen efficiently translocated across the plasma membrane.

Homeoproteins with Intercellular Translocation Properties

Homeoproteins are highly conserved, transactivating transcriptionfactors involved in morphological processes. They bind to DNA through aspecific sequence of 60 amino acids. The DNA-binding homeodomain is themost highly conserved sequence of the homeoprotein. Severalhomeoproteins exhibit PTD like activity; they are capable of efficienttranslocation across cell membranes in an energy-independent andendocytosis-independent manner without cell type specificity.

The Antennapedia protein (Antp) is a trans-activating factor capable oftranslocation across cell membranes; the minimal sequence capable oftranslocation is a 16 amino acid peptide corresponding to the thirdhelix of the protein's homeodomain. The internalization of this helixoccurs at 4° C. suggesting that this process is not endocytosisdependent. Peptides of up to 100 amino acids produced as fusion proteinswith AntpHD penetrate cell membranes.

Other homeodomains capable of translocation include Fushi tarazu (Ftz)and Engrailed (En) homeodomain. Since the third helix of allhomeodomains is highly conserved, it is likely that other homeodomainsmay possess similar characteristics.

Synthetic PTDs

Several PTD peptides have been synthesized. Many of these syntheticpeptides are based on existing and well documented peptides, whileothers are selected for their basic residues and/or positive charge,which are thought to be crucial for PTD function. These syntheticpeptides include: PTD-4 (YARAAARQARA) [SEQ ID NO: 153]; PTD-5(RRQRRTSKLMKR) [SEQ ID NO: 154]; MST-1 (AAVLLPVLLAAR) [SEQ ID NO: 155];L-R9 (RRRRRRRRR) [SEQ ID NO: 156]; and Peptide 2 (SGWFRRWKK) [SEQ ID NO:157].

Human PTDs

Human PTDs may circumvent potential immunogenicity issues when used as atherapeutic upon introduction into a human patient. Peptides with PTDsequences include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX,MOX-2, and FtzPTD, all of which share the sequence found in AntpPTD(RQIKIWFQNRRMKWKK) [SEQ ID NO: 158]. Other PTDs include Islet-1,interleukin-1β, tumor necrosis factor, and the hydrophobic sequence fromKaposi-FGF (K-FGF or FGF-4) signal peptide which is capable of energy-,receptor-, and endocytosis-independent translocation. Unconfirmed PTDsinclude members of the Fibroblast Growth Factor (FGF) family.

At present, it is possible to produce a given protein molecule byrecombinant DNA technology for in vivo therapeutic applications.Although small molecules or peptides capable of crossing cellularmembranes have been successfully designed to deliver small or moderatelylarge proteins, it remains a challenge to deliver the recombinantproteins to desired targets in vivo. Despite developments in the area ofprotein transduction peptides, the classical delivery methods ofprotein-coding genes via adeno-associated virus, adenovirus, lentivirus,herpes virus vectors, and plasmid expression vectors remain thepreferred choice for expression of proteins.

Viral vector-mediated gene expression is considered the most efficientand reliable approach for expressing functional proteins de novo inmitotically active or postmitotically blocked cell types due to theirnatural abilities to deliver the specific genes to permissive cells.Nonetheless, viral vectors invariably are required in large doses toachieve therapeutic expression levels of intended proteins and mayintegrate with the host chromatin material. Because these properties mayhave undesirable consequences for host genetic systems, safety remains aserious concern for their ultimate clinical application.

While an alternative approach, i.e., to produce recombinant proteinsexogenously and then deliver them systemically or by localizedinjections into the target organs, appears to have a better safetyprofile, the delivery and bioavailability of recombinant proteins intocells or tissues needs refinement.

Several studies have shown the potential of PTD in drug discovery andtransduction of proteins up to 120 kDa into different cells. In vivoinjection of fusion proteins systemically has demonstrated theeffectiveness of the PTD in protein delivery. Despite successfulapplications, questions about potency of PTD mediated proteintransduction still remain unsolved. Further, some studies inPTD-mediated fusion protein transduction in vitro/in vivo and attemptingto induce an immune response have failed. Further, intracellularexpression of PTD fusion proteins or other non-secretory proteins maynot achieve the same biodistribution as that of recombinant protein.Further, entry of PTD through the blood-brain barrier remains elusive.

The delivery of a diverse set of cargo ranging from small molecules toparticulate cargo has been attempted using different types of cellpenetrating peptides in vitro and in vivo. However, the internalizationmechanism of these peptides is an unresolved issue to date, withdramatic changes in view regarding the involvement of an energydependent process involving endocytosis as a pathway of internalization.An improvment in the effectiveness of PTDs would significantly increasebioavailability and lower the required doses of existing and noveltherapeutics.

The present invention provides transduction domain peptides that areuseful for the inhibition of kinases. The present invention furtherprovides a class of peptides that include certain transduction domainsthat are useful as inhibitors of kinase activity. The present inventionfurther provides transduction domain peptides that are useful astherapeutic agents for a variety of hyper plastic and neoplasticdisorders. The present invention further provides transduction domainpeptides that are useful as substances to cause cell death.

SUMMARY OF THE INVENTION

The present invention relates to kinase inhibiting compositions and usesthereof and provides isolated kinase inhibiting peptides and usesthereof for inhibiting hyperplasia, for inhibiting the growth ofneoplasms, and for inducing programmed cell death in a cell population.

In one aspect, the present invention provides a method for inhibiting akinase activity of a kinase enzyme, the method comprising the steps: (a)providing a kinase inhibiting composition, wherein the kinase inhibitingcomposition comprises an inhibitory amount of a kinase inhibitingpeptide; (b) contacting kinase enzyme with the kinase inhibitingcomposition of step (a) such that the kinase inhibiting peptideassociates with the kinase enzyme; and (c) inhibiting the kinaseactivity of the kinase enzyme. According to one embodiment of themethod, the kinase inhibiting peptide is a KIP peptide. According toanother embodiment, the kinase inhibiting peptide is acyclin-dependent-kinase inhibitor. According to another embodiment, thekinase inhibiting peptide is a peptide having an amino acid sequenceaccording to Formula IV [Q1-Z1-Z2-Z3-Z4-Z5-Z6-Q2], wherein Q1 and Q2 areindependently absent or present, and wherein if Q1 and Q2 are present,Q1 and Q2 comprise a polypeptide having an amino acid sequence accordingto Formula IV(a) [X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11], wherein X1 is anyamino acid except K, or is absent; X2 is present or is absent; and whenX2 is present, X2 is any amino acid; X3 is any amino acid; X4 is anyamino acid; X5 is any basic amino acid; X6 is any amino acid; X7 is anyamino acid; X8 is any amino acid; X9 is any amino acid; X10 is any aminoacid; X11 is any amino acid, or is absent; and wherein Z1 and Z2 arepresent; wherein Z3 is present or absent, wherein Z4 is absent orpresent, but if Z4 is present, Z3 is present, wherein Z5 is absent orpresent, but if Z5 is present, Z3 and Z4 are present; wherein Z6 isabsent or present, but if Z6 is present, Z3, Z4 and Z5 are present; andeach of Z1, Z2, Z3, Z4, Z5, and Z6, is a peptide selected from the groupconsisting of: (a) X1-X2-B1-B2-X3-B3-X4 [Formula IV(b)], wherein each ofX1, X3 and X4 is a hydrophobic amino acid; X2 is any amino acid; andwherein each of B1, B2 and B3 is a basic amino acid; (b)X1-X2-B1-B2-X3-B3 [Formula IV(c)], wherein each of X1 and X3 is ahydrophobic amino acid, X2 is any amino acid; and wherein each of B1,B2, and B3 is a basic amino acid; (c) X1-X2-B1-B2-X3 [Formula IV(d)],wherein each of X1 and X3 is a hydrophobic amino acid; X2 is any aminoacid; and wherein each of B1 and B2 is a basic amino acid; (d)X1-B1-B2-X2-B3-X3 [Formula IV(e)], wherein X1 is any amino acid; each ofX2 and X3 is a hydrophobic amino acid, and each of B1, B2, and B3 is abasic amino acid; (e) X1-B1-B2-X2-B3 [Formula IV(f)], wherein X1 is ahydrophobic amino acid, H or N; X2 is a hydrophobic amino acid; and eachof B1, B2, and B3 is a basic amino acid; (f) X1-B1-B2-X2 [FormulaIV(g)], wherein X1 is any amino acid; X2 is a hydrophobic amino acid;and each of B1 and B2 is a basic amino acid; (g) X1-X2-B1-B2 [FormulaIV(h)], wherein X1 is a hydrophobic amino acid, X2 is any amino acid;and each of B1 and B2 is a basic amino acid; (h) X1-X2-B1-X3-X4 [FormulaIV(i)], wherein each of X1, X3, and X4 is a hydrophobic amino acid; X2is any amino acid; and B1 is a basic amino acid; (i) X1-B1-X2-X3[Formula IV(j)], wherein X1 is any amino acid; X2 is any amino acid; X3is a hydrophobic amino acid; and B1 is any basic amino acid; (j)X1-X2-B1-X3 [Formula IV(k)], wherein each of X1 and X3 is a hydrophobicamino acid; X2 is any amino acid; and B1 is a basic amino acid; (k)B1-B2-X1-X2-B3-X3 [Formula IV(1)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (1) B1-B2-X1-X2-B3 [Formula IV(m)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (m) B1-B2-X1-X2 [Formula IV(n)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1 and B2 is a basic amino acid; (n)B1-X1-X2-B2-B3-X3 [Formula IV(o)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (o) B1-X1-X2-B2-B3 [Formula IV(p)], wherein each of X1 and X2 is ahydrophobic amino acid, and each of B1, B2, and B3 is a basic aminoacid; and (p) B1-X1-X2-B2 [Formula IV(q)], wherein each of X1 and X2 isa hydrophobic amino acid; and each of B1 and B2 is a basic amino acid;with the proviso that if Q2 is present, the two amino acids immediatelypreceding Q2 as part of Z2, Z3, Z4, Z5, or Z6 cannot be KA. According toone such embodiment, Q1, Z3, Z4, Z5, and Z6 are absent. According toanother such embodiment, X2 in formula IV(a) is a hydrophobic aminoacid, H or N. According to another such embodiment, X2 in formula IV(a)is A. According to another such embodiment, X3 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X3 isselected from the group consisting of L, I, V, and M. According toanother such embodiment, X4 in formula IV(a) is selected from the groupconsisting of Q, A and N. According to another such embodiment, X6 informula IV(a) is selected from the group consisting of Q and N.According to another such embodiment, X7 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X7 informula IV(a) is selected from the group consisting of L, I, V and M.According to another such embodiment, X8 in formula IV(a) is selectedfrom the group consisting of G, A, C, S, T, and Y. According to anothersuch embodiment, X9 in formula IV(a) is a hydrophobic amino acid.According to another such embodiment, X9 in formula IV(a) is selectedfrom the group consisting of L, I, V and M. According to another suchembodiment, X9 in formula IV(a) is V. According to another embodiment ofthe method, the kinase inhibiting peptide is a peptide having an aminoacid sequence selected from the group consisting ofHRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]; WLRRIKAWLRRIKALARQLGVAA [SEQ IDNO: 113]; and WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]. According toanother embodiment of the method, the amino acid sequence of the kinaseinhibiting peptide is HRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]. Accordingto another embodiment of the method, the amino acid sequence of thekinase inhibiting peptide is WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113].According to another embodiment of the method, the amino acid sequenceof the kinase inhibiting peptide is WLRRIKAWLRRALARQLGVA [SEQ ID NO:177]. According to another embodiment of the method, the kinaseinhibiting peptide is a peptide having an amino acid sequence accordingto Formula V [(XXBBXBXX)_(n)] wherein X is any amino acid, B is a basicamino acid, and n is an integer between 2 and 5. According to one suchembodiment, B is K, R or H. According to another embodiment of themethod, the kinase inhibiting peptide is a peptide having an amino acidsequence according to Formula VI [Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2],wherein each of Z1 and Z2 is absent or is a transduction domain; X1 isabsent or present, and if present is selected from the group consistingof A, KA, KKA, KKKA, and RA; X2 is selected from the group consisting ofan aliphatic amino acid, G, L, A, V, I, M, Y, W, and F; X3 is selectedfrom the group consisting of an aliphatic amino acid, V, L, I, A, G, Q,N, S, T, and C; X4 is selected from the group consisting of Q, N, H, Rand K; X5 is selected from the group consisting of Q and N; X6 isselected from the group consisting of an aliphatic amino acid, C, A, G,L, V, I, M, Y, W, and F; X7 is selected from the group consisting of analiphatic amino acid, S, A, C, T, and G; X8 is selected from the groupconsisting of V, L, I, and M; X9 is absent or is any amino acid; and X10is absent or is any amino acid. According to one such embodiment, X2 isselected from the group consisting of G, L, A, V, I, M, Y, W, and F.According to another such embodiment, X3 is selected from the groupconsisting of V, L, I, A, G, Q, N, S, T, and C. According to anothersuch embodiment, X6 is selected from the group consisting of C, A, G, L,V, I, M, Y, W, and F. According to another such embodiment, the kinaseinhibiting peptide is a peptide having an amino acid sequence selectedfrom the group consisting of KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173];FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]; and WLRRIKAWLRRIKALNRQLGVAA [SEQID NO: 142]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is KAFAKLAARLYRKALARQLGVAA[SEQ ID NO: 173]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is FAKLAARLYRKALARQLGVAA [SEQID NO: 163]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is WLRRIKAWLRRIKALNRQLGVAA[SEQ ID NO: 142]. According to another embodiment of the method, thekinase enzyme is a kinase enzyme selected from the group consisting of:Ab 1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI,CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin,CKly1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR,DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit,Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7, GSK, CSK3, Hck,HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK, Insulin R, IRAK,IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR, Lck, LIMK, LIMK1,LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK, Met, Mer, MINK,MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα,PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1,PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI, PKCδ, PKG1, PKG1α, PKG1β, PKR, PLK,PRAK, PTK5, Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4,Rsk/MAPKAP Kinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src,Syk, TAK1, TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3,Ulk2, VRK2, WEE, Yes, ZAP-70 and ZIPK. According to one such embodiment,the kinase enzyme is selected from the group consisting of a ROCKkinase, a Src kinase, a PKC kinase and a Trk kinase. According to somesuch embodiments, the kinase enzyme is a ROCK kinase. According to somesuch embodiments, the kinase enzyme is an Src kinase. According to somesuch embodiments, the kinase enzyme is a PKC kinase. According to somesuch embodiments, the kinase enzyme is a Trk kinase.

In another aspect, the present invention provides a method forinhibiting hyperplasia in a cell population, the method comprising thesteps: (a) providing a therapeutically effective amount of a kinaseinhibiting composition to a subject in need thereof, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide; (b) contacting at least one hyperplastic cell in thecell population with the kinase inhibitory composition such that thekinase inhibiting peptide associates with the at least one hyperplasticcell; and (c) inhibiting the hyperplasia of the at least onehyperplastic cell. According to one embodiment, the kinase inhibitingpeptide is a KIP peptide. According to another embodiment, the kinaseinhibiting peptide is a cyclin-dependent-kinase inhibitor. According toanother embodiment, the kinase inhibiting peptide is a peptide having anamino acid sequence according to Formula IV [Q1-Z1-Z2-Z3-Z4-Z5-Z6-Q2],wherein Q1 and Q2 are independently absent or present, and wherein if Q1and Q2 are present, Q1 and Q2 comprise a polypeptide having an aminoacid sequence according to Formula IV(a)[X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11], wherein X1 is any amino acidexcept K, or is absent; X2 is present or is absent; and when X2 ispresent, X2 is any amino acid; X3 is any amino acid; X4 is any aminoacid; X5 is any basic amino acid; X6 is any amino acid; X7 is any aminoacid; X8 is any amino acid; X9 is any amino acid; X10 is any amino acid;X11 is any amino acid, or is absent; and wherein Z1 and Z2 are present;wherein Z3 is present or absent, wherein Z4 is absent or present, but ifZ4 is present, Z3 is present, wherein Z5 is absent or present, but if Z5is present, Z3 and Z4 are present; wherein Z6 is absent or present, butif Z6 is present, Z3, Z4 and Z5 are present; and each of Z1, Z2, Z3, Z4,Z5, and Z6, is a peptide selected from the group consisting of: (a)X1-X2-B1-B2-X3-B3-X4 [Formula IV(b)], wherein each of X1, X3 and X4 is ahydrophobic amino acid; X2 is any amino acid; and wherein each of B1, B2and B3 is a basic amino acid; (b) X1-X2-B1-B2-X3-B3 [Formula IV(c)],wherein each of X1 and X3 is a hydrophobic amino acid, X2 is any aminoacid; and wherein each of B1, B2, and B3 is a basic amino acid; (c)X1-X2-B1-B2-X3 [Formula IV(d)], wherein each of X1 and X3 is ahydrophobic amino acid; X2 is any amino acid; and wherein each of B1 andB2 is a basic amino acid; (d) X1-B1-B2-X2-B3-X3 [Formula IV(e)], whereinX1 is any amino acid; each of X2 and X3 is a hydrophobic amino acid, andeach of B1, B2, and B3 is a basic amino acid; (e) X1-B1-B2-X2-B3[Formula IV(f)], wherein X1 is a hydrophobic amino acid, H or N; X2 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (f) X1-B1-B2-X2 [Formula IV(g)], wherein X1 is any amino acid; X2is a hydrophobic amino acid; and each of B1 and B2 is a basic aminoacid; (g) X1-X2-B1-B2 [Formula IV(h)], wherein X1 is a hydrophobic aminoacid, X2 is any amino acid; and each of B1 and B2 is a basic amino acid;(h) X1-X2-B1-X3-X4 [Formula IV(i)], wherein each of X1, X3, and X4 is ahydrophobic amino acid; X2 is any amino acid; and B1 is a basic aminoacid; (i) X1-B1-X2-X3 [Formula IV(j)], wherein X1 is any amino acid; X2is any amino acid; X3 is a hydrophobic amino acid; and B1 is a basicamino acid; (j) X1-X2-B1-X3 [Formula IV(k)], wherein each of X1 and X3is a hydrophobic amino acid; X2 is any amino acid; and B1 is a basicamino acid; (k) B1-B2-X1-X2-B3-X3 [Formula IV(1)], wherein each of X1,X2, and X3 is a hydrophobic amino acid; and each of B1, B2, and B3 is abasic amino acid; (1) B1-B2-X1-X2-B3 [Formula IV(m)], wherein each of X1and X2 is a hydrophobic amino acid; and each of B1, B2, and B3 is abasic amino acid; (m) B1-B2-X1-X2 [Formula IV(n)], wherein each of X1and X2 is a hydrophobic amino acid; and each of B1 and B2 is a basicamino acid; (n) B1-X1-X2-B2-B3-X3 [Formula IV(o)], wherein each of X1,X2, and X3 is a hydrophobic amino acid; and each of B1, B2, and B3 is abasic amino acid; (o) B1-X1-X2-B2-B3 [Formula IV(p)], wherein each of X1and X2 is a hydrophobic amino acid, and each of B1, B2, and B3 is abasic amino acid; and (p) B1-X1-X2-B2 [Formula IV(q)], wherein each ofX1 and X2 is a hydrophobic amino acid; and each of B1 and B2 is a basicamino acid; with the proviso that if Q2 is present, the two amino acidsimmediately preceding Q2 as part of Z2, Z3, Z4, Z5, or Z6 cannot be KA.According to one such embodiment, Q1, Z3, Z4, Z5, and Z6 are absent.According to another such embodiment, X2 in formula IV(a) is ahydrophobic amino acid, H or N. According to another such embodiment, X2in formula IV(a) is A. According to another such embodiment, X3 informula IV(a) is a hydrophobic amino acid. According to another suchembodiment, X3 in formula IV(a) is selected from the group consisting ofL, I, V, and M. According to another such embodiment, X4 in formulaIV(a) is selected from the group consisting of Q, A and N.

According to another such embodiment, X6 in formula IV(a) is selectedfrom the group consisting of Q and N. According to another suchembodiment, X7 in formula IV(a) is a hydrophobic amino acid. Accordingto another such embodiment, X7 in formula IV(a) is selected from thegroup consisting of L, I, V and M. According to another such embodiment,X8 in formula IV(a) is selected from the group consisting of G, A, C, S,T, and Y. According to another such embodiment, X9 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X9 informula IV(a) is selected from the group consisting of L, I, V and M.According to another such embodiment, in formula IV(a) X9 is V.According to another embodiment of the method, the kinase inhibitingpeptide is a peptide having an amino acid sequence selected from thegroup consisting of HRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166];WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113]; and WLRRIKAWLRRALARQLGVA [SEQID NO: 177]. According to another embodiment of the method, the aminoacid sequence of the kinase inhibiting peptide is HRRIKAWLKKILALARQLGVAA[SEQ ID NO: 166]. According to another embodiment of the method, theamino acid sequence of the kinase inhibiting peptide isWLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113]. According to anotherembodiment of the method, the amino acid sequence of the kinaseinhibiting peptide is WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]. Accordingto another embodiment of the method, the kinase inhibiting peptide is apeptide having an amino acid sequence according to Formula V[(XXBBXBXX)_(n)] wherein X is any amino acid, B is a basic amino acid,and n is an integer between 2 and 5. According to one such embodiment, Bis K, R or H. According to another embodiment of the method, the kinaseinhibiting peptide is a peptide having an amino acid sequence accordingto Formula VI [Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2], wherein each of Z1and Z2 is absent or is a transduction domain; X1 is absent or present,and if present is selected from the group consisting of A, KA, KKA,KKKA, and RA; X2 is selected from the group consisting of an aliphaticamino acid, G, L, A, V, I, M, Y, W, and F; X3 is selected from the groupconsisting of an aliphatic amino acid, V, L, I, A, G, Q, N, S, T, and C;X4 is selected from the group consisting of Q, N, H, R and K; X5 isselected from the group consisting of Q and N; X6 is selected from thegroup consisting of an aliphatic amino acid, C, A, G, L, V, I, M, Y, W,and F; X7 is selected from the group consisting of an aliphatic aminoacid, S, A, C, T, and G; X8 is selected from the group consisting of V,L, I, and M; X9 is absent or is any amino acid; and X10 is absent or isany amino acid. According to one such embodiment, X2 is selected fromthe group consisting of G, L, A, V, I, M, Y, W, and F. According toanother such embodiment, X3 is selected from the group consisting of V,L, I, A, G, Q, N, S, T, and C. According to another such embodiment, X6is selected from the group consisting of C, A, G, L, V, I, M, Y, W, andF. According to another such embodiment, the kinase inhibiting peptideis a peptide having an amino acid sequence selected from the groupconsisting of KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173];FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]; and WLRRIKAWLRRIKALNRQLGVAA [SEQID NO: 142]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is KAFAKLAARLYRKALARQLGVAA[SEQ ID NO: 173]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is FAKLAARLYRKALARQLGVAA [SEQID NO: 163]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is WLRRIKAWLRRIKALNRQLGVAA[SEQ ID NO: 142].

In another aspect, the present invention provides a method forinhibiting growth of a neoplasm, the method comprising the steps: (a)providing a therapeutically effective amount of a kinase inhibitingcomposition to a subject in need thereof, wherein the kinase inhibitingcomposition comprises an inhibitory amount of a kinase inhibitingpeptide; and (b) contacting the neoplasm with the kinase inhibitingcomposition such that the kinase inhibiting peptide associates with theneoplasm; and (c) inhibiting the growth of the neoplasm. According toone embodiment, the kinase inhibiting peptide is a KIP peptide.According to another embodiment, the kinase inhibiting peptide is acyclin-dependent-kinase inhibitor. According to another embodiment, thekinase inhibiting peptide is a peptide having an amino acid sequenceaccording to Formula IV [Q1-Z1-Z2-Z3-Z4-Z5-Z6-Q2], wherein Q1 and Q2 areindependently absent or present, and wherein if Q1 and Q2 are present,Q1 and Q2 comprise a polypeptide having an amino acid sequence accordingto Formula IV(a) [X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11], wherein X1 is anyamino acid except K, or is absent; X2 is present or is absent; and whenX2 is present, X2 is any amino acid; X3 is any amino acid; X4 is anyamino acid; X5 is any basic amino acid; X6 is any amino acid; X7 is anyamino acid; X8 is any amino acid; X9 is any amino acid; X10 is any aminoacid; X11 is any amino acid, or is absent; and wherein Z1 and Z2 arepresent; wherein Z3 is present or absent, wherein Z4 is absent orpresent, but if Z4 is present, Z3 is present, wherein Z5 is absent orpresent, but if Z5 is present, Z3 and Z4 are present; wherein Z6 isabsent or present, but if Z6 is present, Z3, Z4 and Z5 are present; andeach of Z1, Z2, Z3, Z4, Z5, and Z6, is a peptide selected from the groupconsisting of: (a) X1-X2-B1-B2-X3-B3-X4 [Formula IV(b)], wherein each ofX1, X3 and X4 is a hydrophobic amino acid; X2 is any amino acid; andwherein each of B1, B2 and B3 is a basic amino acid; (b)X1-X2-B1-B2-X3-B3 [Formula IV(c)], wherein each of X1 and X3 is ahydrophobic amino acid, X2 is any amino acid; and wherein each of B1,B2, and B3 is a basic amino acid; (c) X1-X2-B1-B2-X3 [Formula IV(d)],wherein each of X1 and X3 is a hydrophobic amino acid; X2 is any aminoacid; and wherein each of B1 and B2 is a basic amino acid; (d)X1-B1-B2-X2-B3-X3 [Formula IV(e)], wherein X1 is any amino acid; each ofX2 and X3 is a hydrophobic amino acid, and each of B1, B2, and B3 is abasic amino acid; (e) X1-B1-B2-X2-B3 [Formula IV(f)], wherein X1 is ahydrophobic amino acid, H or N; X2 is a hydrophobic amino acid; and eachof B1, B2, and B3 is a basic amino acid; (f) X1-B1-B2-X2 [FormulaIV(g)], wherein X1 is any amino acid; X2 is a hydrophobic amino acid;and each of B1 and B2 is a basic amino acid; (g) X1-X2-B1-B2 [FormulaIV(h)], wherein X1 is a hydrophobic amino acid, X2 is any amino acid;and each of B1 and B2 is a basic amino acid; (h) X1-X2-B1-X3-X4 [FormulaIV(i)], wherein each of X1, X3, and X4 is a hydrophobic amino acid; X2is any amino acid; and B1 is a basic amino acid; (i) X1-B1-X2-X3[Formula IV(j)], wherein X1 is any amino acid; X2 is any amino acid; X3is a hydrophobic amino acid; and B1 is a basic amino acid; (j)X1-X2-B1-X3 [Formula IV(k)], wherein each of X1 and X3 is a hydrophobicamino acid; X2 is any amino acid; and B1 is a basic amino acid; (k)B1-B2-X1-X2-B3-X3 [Formula IV(1)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (1) B1-B2-X1-X2-B3 [Formula IV(m)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (m) B1-B2-X1-X2 [Formula IV(n)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1 and B2 is a basic amino acid; (n)B1-X1-X2-B2-B3-X3 [Formula IV(o)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (o) B1-X1-X2-B2-B3 [Formula IV(p)], wherein each of X1 and X2 is ahydrophobic amino acid, and each of B1, B2, and B3 is a basic aminoacid; and (p) B1-X1-X2-B2 [Formula IV(q)], wherein each of X1 and X2 isa hydrophobic amino acid; and each of B1 and B2 is a basic amino acid;with the proviso that if Q2 is present, the two amino acids immediatelypreceding Q2 as part of Z2, Z3, Z4, Z5, or Z6 cannot be KA. According tosuch embodiment, Q1, Z3, Z4, Z5, and Z6 are absent. According to anothersuch embodiment, X2 in formula IV(a) is a hydrophobic amino acid, H orN. According to another such embodiment, X2 in formula IV(a) is A.According to another such embodiment, X3 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X3 informula IV(a) is selected from the group consisting of L, I, V, and M.According to another such embodiment, X4 in formula IV(a) is selectedfrom the group consisting of Q, A and N. According to another suchembodiment, X6 in formula IV(a) is selected from the group consisting ofQ and N. According to another such embodiment, X7 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X7 informula IV(a) is selected from the group consisting of L, I, V and M.According to another such embodiment, X8 in formula IV(a) is selectedfrom the group consisting of G, A, C, S, T, and Y. According to anothersuch embodiment, X9 in formula IV(a) is a hydrophobic amino acid.According to another such embodiment, X9 in formula IV(a) is selectedfrom the group consisting of L, I, V and M. According to another suchembodiment, X9 in formula IV(a) is V. According to another embodiment ofthe method, the kinase inhibiting peptide is a peptide having an aminoacid sequence selected from the group consisting ofHRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]; WLRRIKAWLRRIKALARQLGVAA [SEQ IDNO: 113]; and WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]. According toanother embodiment of the method, the amino acid sequence of the kinaseinhibiting peptide is HRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]. Accordingto another embodiment of the method, the amino acid sequence of thekinase inhibiting peptide is WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113].According to another embodiment of the method, the amino acid sequenceof the kinase inhibiting peptide is WLRRIKAWLRRALARQLGVA [SEQ ID NO:177]. According to another embodiment of the method, the kinaseinhibiting peptide is a peptide having an amino acid sequence accordingto Formula V [(XXBBXBXX)_(n)] wherein X is any amino acid, B is a basicamino acid, and n is an integer between 2 and 5. According to one suchembodiment, B is K, R or H. According to another embodiment of themethod, the kinase inhibiting peptide is a peptide having an amino acidsequence according to Formula VI [Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2],wherein each of Z1 and Z2 is absent or is a transduction domain; X1 isabsent or present, and if present is selected from the group consistingof A, KA, KKA, KKKA, and RA; X2 is selected from the group consisting ofan aliphatic amino acid, G, L, A, V, I, M, Y, W, and F; X3 is selectedfrom the group consisting of an aliphatic amino acid, V, L, I, A, G, Q,N, S, T, and C; X4 is selected from the group consisting of Q, N, H, Rand K; X5 is selected from the group consisting of Q and N; X6 isselected from the group consisting of an aliphatic amino acid, C, A, G,L, V, I, M, Y, W, and F; X7 is selected from the group consisting of analiphatic amino acid, S, A, C, T, and G; X8 is selected from the groupconsisting of V, L, I, and M; X9 is absent or is any amino acid; and X10is absent or is any amino acid. According to one such embodiment, X2 isselected from the group consisting of G, L, A, V, I, M, Y, W, and F.According to another such embodiment, X3 is selected from the groupconsisting of V, L, I, A, G, Q, N, S, T, and C. According to anothersuch embodiment, X6 is selected from the group consisting of C, A, G, L,V, I, M, Y, W, and F. According to another embodiment of the method, thekinase inhibiting peptide is a peptide having an amino acid sequenceselected from the group consisting of KAFAKLAARLYRKALARQLGVAA [SEQ IDNO: 173]; FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]; andWLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142]. According to anotherembodiment of the method, the amino acid sequence of the kinaseinhibiting peptide is KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173].According to another embodiment of the method, the amino acid sequenceof the kinase inhibiting peptide is FAKLAARLYRKALARQLGVAA [SEQ ID NO:163]. According to another embodiment of the method, the amino acidsequence of the kinase inhibiting peptide is WLRRIKAWLRRIKALNRQLGVAA[SEQ ID NO: 142]. According to another embodiment of the method, theneoplasm is a neoplasm selected from the group consisting of apapilloma, an adenoma, a hydatidiform mole, a fibroma, a chondroma, anosteoma, a leiomyoma, a rhabdomyoma, a lipoma, a hemangioma, alymphangioma, a polycythemia vera, an infectious mononucleosis, a“benign” glioma, a meningioma, a ganglioneuroma, a neurilemmoma, aneurofibroma, a pigmented nevus (mole), a pheochromocytoma, a carcinoidtumors, a teratoma, a carcinoma, an adenocarcinoma, a basal cellcarcinoma, a choriocarcinoma, a fibrosarcoma, a chondrosarcoma, anosteosarcoma, a leiomyosarcoma, a rhabdomyosarcoma, a liposarcoma, ahemangiosarcoma, a lymphangiosarcoma, a myelocytic leukemia, anerythrocytic leukemia, a lymphocytic leukemia, a multiple myeloma, amonocytic leukemia, an Ewing's sarcoma, a non-Hodgkin's malignantlymphoma, a medulloblastoma, an oligodendroglioma, a neurilemmal sarcomamalignant melanoma, thymoma, a glioblastoma multiforme, an astrocytoma,an ependymoma, an meningeal sarcoma, a neuroblastoma (schwannoma), aneurofibrosarcoma, a malignant pheochromocytoma, a retinoblastoma, acarcinoid tumor, a nephroblastoma (Wilms' tumor), a teratocarcinoma andan embryonal carcinoma with choriocarcinoma.

In another aspect, the present invention provides a method for inducingprogrammed cell death in a cell population, the method comprising thesteps: (a) providing a kinase inhibiting composition, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide; (b) contacting at least one cell in the cellpopulation with the kinase inhibiting composition such that the kinaseinhibiting peptide associates with the at least one cell; and (c)inducing programmed cell death of the at least one cell. According toone embodiment of the method, the kinase inhibiting peptide is a KIPpeptide. According to another embodiment, the kinase inhibiting peptideis a cyclin-dependent-kinase inhibitor. According to another embodiment,the kinase inhibiting peptide is a peptide having an amino acid sequenceaccording to Formula IV [Q1-Z1-Z2-Z3-Z4-Z5-Z6-Q2], wherein Q1 and Q2 areindependently absent or present, and wherein if Q1 and Q2 are present,Q1 and Q2 comprise a polypeptide having an amino acid sequence accordingto Formula IV(a) [X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11], wherein X1 is anyamino acid except K, or is absent; X2 is present or is absent; and whenX2 is present, X2 is any amino acid; X3 is any amino acid; X4 is anyamino acid; X5 is any basic amino acid; X6 is any amino acid; X7 is anyamino acid; X8 is any amino acid; X9 is any amino acid; X10 is any aminoacid; X11 is any amino acid, or is absent; and wherein Z1 and Z2 arepresent; wherein Z3 is present or absent, wherein Z4 is absent orpresent, but if Z4 is present, Z3 is present, wherein Z5 is absent orpresent, but if Z5 is present, Z3 and Z4 are present; wherein Z6 isabsent or present, but if Z6 is present, Z3, Z4 and Z5 are present; andeach of Z1, Z2, Z3, Z4, Z5, and Z6, is a peptide selected from the groupconsisting of: (a) X1-X2-B1-B2-X3-B3-X4 [Formula IV(b)], wherein each ofX1, X3 and X4 is a hydrophobic amino acid; X2 is any amino acid; andwherein each of B1, B2 and B3 is a basic amino acid; (b)X1-X2-B1-B2-X3-B3 [Formula IV(c)], wherein each of X1 and X3 is ahydrophobic amino acid, X2 is any amino acid; and wherein each of B1,B2, and B3 is a basic amino acid; (c) X1-X2-B1-B2-X3 [Formula IV(d)],wherein each of X1 and X3 is a hydrophobic amino acid; X2 is any aminoacid; and wherein each of B1 and B2 is a basic amino acid; (d)X1-B1-B2-X2-B3-X3 [Formula IV(e)], wherein X1 is any amino acid; each ofX2 and X3 is a hydrophobic amino acid, and each of B1, B2, and B3 is abasic amino acid; (e) X1-B1-B2-X2-B3 [Formula IV(f)], wherein X1 is ahydrophobic amino acid, H or N; X2 is a hydrophobic amino acid; and eachof B1, B2, and B3 is a basic amino acid; (f) X1-B1-B2-X2 [FormulaIV(g)], wherein X1 is any amino acid; X2 is a hydrophobic amino acid;and each of B1 and B2 is a basic amino acid; (g) X1-X2-B1-B2 [FormulaIV(h)], wherein X1 is a hydrophobic amino acid, X2 is any amino acid;and each of B1 and B2 is a basic amino acid; (h) X1-X2-B1-X3-X4 [FormulaIV(i)], wherein each of X1, X3, and X4 is a hydrophobic amino acid; X2is any amino acid; and B1 is a basic amino acid; (i) X1-B1-X2-X3[Formula IV(j)], wherein X1 is any amino acid; X2 is any amino acid; X3is a hydrophobic amino acid; and B1 is a basic amino acid; (j)X1-X2-B1-X3 [Formula IV(k)], wherein each of X1 and X3 is a hydrophobicamino acid; X2 is any amino acid; and B1 is a basic amino acid; (k)B1-B2-X1-X2-B3-X3 [Formula IV(1)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (1) B1-B2-X1-X2-B3 [Formula IV(m)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (m) B1-B2-X1-X2 [Formula IV(n)], wherein each of X1 and X2 is ahydrophobic amino acid; and each of B1 and B2 is a basic amino acid; (n)B1-X1-X2-B2-B3-X3 [Formula IV(o)], wherein each of X1, X2, and X3 is ahydrophobic amino acid; and each of B1, B2, and B3 is a basic aminoacid; (o) B1-X1-X2-B2-B3 [Formula IV(p)], wherein each of X1 and X2 is ahydrophobic amino acid, and each of B1, B2, and B3 is a basic aminoacid; and (p) B1-X1-X2-B2 [Formula IV(q)], wherein each of X1 and X2 isa hydrophobic amino acid; and each of B1 and B2 is any basic amino acid;with the proviso that if Q2 is present, the two amino acids immediatelypreceding Q2 as part of Z2, Z3, Z4, Z5, or Z6 cannot be KA. According toone such embodiment, Q1, Z3, Z4, Z5, and Z6 are absent. According toanother such embodiment, X2 in formula IV(a) is a hydrophobic aminoacid, H or N. According to another such embodiment, X2 in formula IV(a)is A. According to another such embodiment, X3 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X3 informula IV(a) is selected from the group consisting of L, I, V, and M.According to another such embodiment, X4 in formula IV(a) is selectedfrom the group consisting of Q, A and N. According to another suchembodiment, X6 in formula IV(a) is selected from the group consisting ofQ and N. According to another such embodiment, X7 in formula IV(a) is ahydrophobic amino acid. According to another such embodiment, X7 informula IV(a) is selected from the group consisting of L, I, V and M.According to another such embodiment, X8 in formula IV(a) is selectedfrom the group consisting of G, A, C, S, T, and Y. According to anothersuch embodiment, X9 in formula IV(a) is a hydrophobic amino acid.According to another such embodiment, X9 in formula IV(a) is selectedfrom the group consisting of L, I, V and M. According to another suchembodiment, X9 in formula IV(a) is V. According to another embodiment ofthe method, the kinase inhibiting peptide is a peptide having an aminoacid sequence selected from the group consisting ofHRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]; WLRRIKAWLRRIKALARQLGVAA [SEQ IDNO: 113]; and WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]. According toanother embodiment of the method, the amino acid sequence of the kinaseinhibiting peptide is HRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]. Accordingto another embodiment of the method, the amino acid sequence of thekinase inhibiting peptide is WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113].According to another embodiment of the method, the amino acid sequenceof the kinase inhibiting peptide is WLRRIKAWLRRALARQLGVA [SEQ ID NO:177]. According to another embodiment of the method, the kinaseinhibiting peptide is a peptide having an amino acid sequence accordingto Formula V [(XXBBXBXX)_(n)] wherein X is any amino acid, B is a basicamino acid, and n is an integer between 2 and 5. According to one suchembodiment, B is K, R or H. According to another embodiment of themethod, the kinase inhibiting peptide is a peptide having an amino acidsequence according to Formula VI [Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2],wherein each of Z1 and Z2 is absent or is a transduction domain; X1 isabsent or present, and if present is selected from the group consistingof A, KA, KKA, KKKA, and RA; X2 is selected from the group consisting ofan aliphatic amino acid, G, L, A, V, I, M, Y, W, and F; X3 is selectedfrom the group consisting of an aliphatic amino acid, V, L, I, A, G, Q,N, S, T, and C; X4 is selected from the group consisting of Q, N, H, Rand K; X5 is selected from the group consisting of Q and N; X6 isselected from the group consisting of an aliphatic amino acid, C, A, G,L, V, I, M, Y, W, and F; X7 is selected from the group consisting of analiphatic amino acid, S, A, C, T, and G; X8 is selected from the groupconsisting of V, L, I, and M; X9 is absent or is any amino acid; and X10is absent or is any amino acid. According to one such embodiment, X2 isselected from the group consisting of G, L, A, V, I, M, Y, W, and F.According to another such embodiment, X3 is selected from the groupconsisting of V, L, I, A, G, Q, N, S, T, and C. According to anothersuch embodiment, X6 is selected from the group consisting of C, A, G, L,V, I, M, Y, W, and F. According to another such embodiment, the kinaseinhibiting peptide is a peptide having an amino acid sequence selectedfrom the group consisting of KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173];FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]; and WLRRIKAWLRRIKALNRQLGVAA [SEQID NO: 142]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is KAFAKLAARLYRKALARQLGVAA[SEQ ID NO: 173]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is FAKLAARLYRKALARQLGVAA [SEQID NO: 163]. According to another such embodiment, the amino acidsequence of the kinase inhibiting peptide is WLRRIKAWLRRIKALNRQLGVAA[SEQ ID NO: 142]. According to another embodiment of the method, thecell is a prokaryotic cell. According to another embodiment of themethod, the cell is a eukaryotic cell. According to another embodimentof the method, the programmed cell death occurs by apoptosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows MCF-7 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 15 μM.

FIG. 2 shows MDA 231 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 20 μM.

FIG. 3 shows SF539 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 27 μM.

FIG. 4 shows HT29 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 27 μM.

FIG. 5 shows Paca 2 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 43 μM.

FIG. 6 shows PC3 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 36 μM.

FIG. 7 shows A549 cells treated with different doses of four KIPpeptides. Peptide 1 is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166], peptide2 is WLRRIKAHRRIKALARQLGVAA [SEQ ID NO: 167], peptide 3 is WLRRIKAWLRR[SEQ ID NO: 168], and peptide 4 is WLRRIKAWLRRALNRQLGVAA [SEQ ID NO:169]. For all peptides, the IC50 concentration was below 35 μM.

FIG. 8 shows that KIP peptides induce apoptosis in a cancer cell line.MCF-7 cells were treated for 24 hours with 3 μM (top panels) or 10 μM(bottom panels) of KIP peptide HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166].The cells were stained with Hoescht dye (panels A and D; blue), AnnexinV antibody (panels B and E; green), and propidium iodide (panels C andF; red). For both concentrations tested, annexin V staining was muchmore intense than propidium iodide staining indicating that KIP peptidescan induce apoptosis in a cancer cell line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides therapeutic kinase inhibitingcompositions and methods of use thereof.

The terms “administering” or “administration” as used herein are usedinterchangeably and include in vivo administration, as well asadministration directly to tissue ex vivo. Generally, compositions maybe administered systemically either orally, buccally, parenterally,topically, by inhalation or insufflation (i.e., through the mouth orthrough the nose), or rectally in dosage unit formulations containingthe conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired, or may be locally administered bymeans such as, but not limited to, injection, implantation, grafting,topical application, or parenterally. The term “parenteral” as usedherein refers to introduction into the body by way of an injection(i.e., administration by injection), including, for example,subcutaneously (i.e., an injection beneath the skin), intramuscularly(i.e., an injection into a muscle), intravenously (i.e., an injectioninto a vein), intrathecally (i.e., an injection inot the space aroundthe spinal cord or under the arachnoid membrane of the brain),intrasternal injection or infusion techniques. A parenterallyadministered composition is delivered using a needle, e.g., a surgicalneedle. The term “surgical needle” as used herein, refers to any needleadapted for delivery of fluid (i.e., capable of flow) compositions intoa selected anatomical structure. Injectable preparations, such assterile injectable aqueous or oleaginous suspensions, may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents.

Additional administration may be performed, for example, intravenously,pericardially, orally, via implant, transmucosally, transdermally,intramuscularly, subcutaneously, intraperitoneally, intrathecally,intralymphatically, intralesionally, or epidurally. Administering can beperformed, for example, once, a plurality of times, and/or over one ormore extended periods. The term “topical administration” and “topicallyapplying” as used herein are used interchangeably to refer to deliveringa peptide, the nucleic acid, or a vector comprising the peptide or thenucleic acid onto one or more surfaces of a tissue or cell, includingepithelial surfaces.'

Although topical administration, in contrast to transdermaladministration, generally provides a local rather than a systemiceffect. The terms “topical administration” and “transdermaladministration” as used herein, unless otherwise stated or implied, areused interchangeably.

The term “associate” or “associates” as used herein refers to joining,connecting, or combining to, either directly, indirectly, actively,inactively, inertly, non-inertly, completely or incompletely.

The abbreviations used herein for amino acids are those abbreviationswhich are conventionally used: A=Ala=Alanine; R=Arg=Arginine;N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine;E=Glu=Gutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=Isoleucine;L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine; F=Phe=Phenyalanine;P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan;Y=Tyr=Tyrosine; V=Val=Valine. The amino acids may be L- or D-aminoacids. An amino acid may be replaced by a synthetic amino acid which isaltered so as to increase the half-life of the peptide or to increasethe potency of the peptide, or to increase the bioavailability of thepeptide.

The term “contacting” as used herein refers to bring or put in contact,to be in or come into contact. The term “contact” as used herein refersto a state or condition of touching or of immediate or local proximity.Contacting a composition to a target destination, such as, but notlimited to, an organ, tissue, cell, or tumor, may occur by any means ofadministration known to the skilled artisan.

Methods exist for the transduction and the transfection of nucleic acidsinto cells. The terms “transduction,” or “transduce” as used herein areused interchangeably to refer to the process of crossing biologicalmembranes. The crossing of biological membranes may be from one cell toanother, from the extracellular environment to the intracellularenvironment, or across a cell membrane or nuclear membrane. Materialsthat may undergo transduction include, but are not limited to, proteins,fusion proteins, peptides, polypeptides, amino acids, viral DNA, andbacterial DNA.

The term “regulatory sequence” (also referred to as a “regulatoryregion” or “regulatory element”) refers to a promoter, enhancer or othersegment of DNA where regulatory proteins, such as transcription factors,bind preferentially to control gene expression and thus proteinexpression.

The term “controllable regulatory element” as used herein refers tonucleic acid sequences capable of effecting the expression of thenucleic acids, or the peptide or protein product thereof. Controllableregulatory elements may be operably linked to the nucleic acids,peptides, or proteins of the present invention. The controllableregulatory elements, such as, but not limited to, control sequences,need not be contiguous with the nucleic acids, peptides, or proteinswhose expression they control as long as they function to direct theexpression thereof. Thus, for example, intervening untranslated yettranscribed sequences may be present between a promoter sequence and anucleic acid of the present invention and the promoter sequence maystill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, termination signals, and ribosome binding sites.

The term “hybridization” refers to the binding of two single strandednucleic acid molecules to each other through base pairing. Nucleotideswill bind to their complement under normal conditions, so two perfectlycomplementary strands will bind (or ‘anneal’) to each other readily.However, due to the different molecular geometries of the nucleotides, asingle inconsistency between the two strands will make binding betweenthem more energetically unfavorable. The effects of base incompatibilitymay be measured by quantifying the rate at which two strands anneal,this may provide information as to the similarity in base sequencebetween the two strands being annealed.

The term “isolated” refers to material, such as a nucleic acid, apeptide, or a protein, which is: (1) substantially or essentially freefrom components that normally accompany or interact with it as found inits naturally occurring environment. The terms “substantially oressentially free” are used to refer to a material, which is at least 80%free from components that normally accompany or interact with it asfound in its naturally occurring environment. The isolated materialoptionally comprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a location in thecell (e.g., genome or subcellular organelle) not native to a materialfound in that environment. The alteration to yield the syntheticmaterial may be performed on the material within, or removed, from itsnatural state. For example, a naturally occurring nucleic acid becomesan isolated nucleic acid if it is altered, or if it is transcribed fromDNA that has been altered, by means of human intervention performedwithin the cell from which it originates. See, for example, Compoundsand Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec,U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting inEukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturallyoccurring nucleic acid (for example, a promoter) becomes isolated if itis introduced by non-naturally occurring means to a locus of the genomenot native to that nucleic acid. Nucleic acids that are “isolated” asdefined herein also are referred to as “heterologous” nucleic acids.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect may include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect may also include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.The term “therapeutically effective amount” or an “amount effective” ofone or more of the active agents of the present invention is an amountthat is sufficient to provide a therapeutic effect. Generally, aneffective amount of the active agents that can be employed ranges fromabout 0.000001 mg/kg body weight to about 100 mg/kg body weight.However, dosage levels are based on a variety of factors, including thetype of injury, the age, weight, sex, medical condition of the patient,the severity of the condition, the route of administration, and theparticular active agent employed. Thus the dosage regimen may varywidely, but can be determined routinely by a physician using standardmethods.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, composition or other substance that provides atherapeutic effect. The term “active” as used herein refers to theingredient, component or constituent of the compositions of the presentinvention responsible for the intended therapeutic effect. The terms“therapeutic agent” and “active agent” are used interchangeably herein.

The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e., dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED50 whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The term “drug” as used herein refers to a therapeutic agent or anysubstance, other than food, used in the prevention, diagnosis,alleviation, treatment, or cure of disease.

The term “reduce” or “reducing” as used herein refers to limitoccurrence of the disorder in individuals at risk of developing thedisorder.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The terms “inhibiting”, “inhibit” or “inhibition” as used herein areused to refer to reducing the amount or rate of a process, to stoppingthe process entirely, or to decreasing, limiting, or blocking the actionor function thereof. Inhibition may include a reduction or decrease ofthe amount, rate, action function, or process by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% when compared to areference substance, wherein the reference substance is a substance thatis not inhibited.

The term “kinase” as used herein refers to a type of enzyme thattransfers phosphate groups from high-energy donor molecules to specifictarget molecules or substrates. High-energy donor groups may include,but are not limited, to ATP.

The term “kinase inhibiting peptide” (or “KIP”) as used herein refers toan amino acid sequence comprising a KIP amino acid sequence. A KIP aminoacid sequence within a peptide or peptidomimetic conveys to the peptideor peptidomimetic certain kinase inhibiting capabilities. KIP1, KIP2 andKIP3 are classes of KIP amino acid sequences.

The term “peptidomimetic” as used herein refers to a small protein-likechain designed to mimic a peptide. A peptidomimetic typically arisesfrom modification of an existing peptide in order to alter themolecule's properties.

The term “kinase substrate” as used herein refers to a substrate thatcan be phosphorylated by a kinase.

The term “kinase activity” as used herein refers to kinase mediatedphosphorylation of a kinase substrate.

The term “mammalian cell” as used herein refers to a cell derived froman animal of the class Mammalia. As used herein, mammalian cells mayinclude normal, abnormal and transformed cells. Examples of mammaliancells utilized within the present invention, include, but are notlimited to, neurons, epithelial cells, muscle cells, blood cells, immunecells, stem cells, osteocytes, endothelial cells and blast cells.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

The term “operably linked” refers to a functional linkage between apromoter and a second sequence, wherein the promoter sequence initiatesand mediates transcription of the DNA sequence corresponding to thesecond sequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, are contiguous and in the same reading frame.

The term “polynucleotide” refers to a deoxyribopolynucleotide,ribopolynucleotide, or analogs thereof that have the essential nature ofa natural ribonucleotide in that they hybridize, under stringenthybridization conditions, to substantially the same nucleotide sequenceas naturally occurring nucleotides and/or allow translation into thesame amino acid(s) as the naturally occurring nucleotide(s). Apolynucleotide may be full-length or a subsequence of a native orheterologous structural or regulatory gene. Unless otherwise indicated,the term includes reference to the specified sequence as well as thecomplementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The term “peptide” as used herein refers to a polypeptide, protein orpeptidomimetic. The terms “polypeptide”, “peptide” and “protein” areused herein to refer to a polymer of amino acid residues. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theessential nature of such analogues of naturally occurring amino acids isthat, when incorporated into a protein that protein is specificallyreactive to antibodies elicited to the same protein but consistingentirely of naturally occurring amino acids. The terms “polypeptide”,“peptide” and “protein” also are inclusive of modifications including,but not limited to, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation. It will be appreciated, as is well known and as notedabove, that polypeptides may not be entirely linear. For instance,polypeptides may be branched as a result of ubiquitination, and they maybe circular, with or without branching, generally as a result ofposttranslational events, including natural processing event and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods, aswell.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

The term “nucleotide” refers to a chemical compound that consists of aheterocyclic base, a sugar, and one or more phosphate groups. In themost common nucleotides the base is a derivative of purine orpyrimidine, and the sugar is the pentose deoxyribose or ribose.Nucleotides are the monomers of nucleic acids, with three or morebonding together in order to form a nucleic acid. Nucleotides are thestructural units of RNA, DNA, and several cofactors, including, but notlimited to, CoA, FAD, DMN, NAD, and NADP. The purines include adenine(A), and guanine (G); the pyrimidines include cytosine (C), thymine (T),and uracil (U).

The term “reduce” or “reducing” as used herein refers to a decrease insize or the slowing of the growth or proliferative rate of a cell of aneoplasm or hyperplasia.

The term “neoplasia” as used herein refers to the abnormal proliferationof cells that results in a neoplasm. The term “neoplasm” as used hereinrefers to an abnormal mass of tissue, the growth of which exceeds and isuncoordinated with that of the normal tissues, and persists in the sameexcessive manner after the cessation of the stimulus which evoked thechange. A neoplasm can be benign, potentially malignant, or malignant.Benign neoplasms include uterine fibroids and melanocytic nevi (skinmoles). These neoplasms do not transform into cancer. Potentiallymalignant neoplasms include carcinoma in situ. These neoplasms do notinvade and destroy but, given enough time, will transform into a cancer.Malignant neoplasms are commonly called cancer. These neoplasms invadeand destroy the surrounding tissue, may form metastases and eventuallykill the subject.

The term “hyperplasia” as used herein is a general term referring to theproliferation of cells within an organ or tissue beyond that which isordinarily seen in e.g. constantly dividing cells. Hyperplasia mayresult in the gross enlargement of an organ, the formation of a benigntumor, or may be visible only under a microscope. Hyperplasia isconsidered to be a physiological response to a specific stimulus, andthe cells of a hyperplastic growth remain subject to normal regulatorycontrol mechanisms. Hyperplasia may be in or on a subject. Hyperplasiamay be due to any number of causes, including increased demand, chronicinflammatory response, hormonal dysfunctions, or compensation for damageor disease elsewhere. Hyperplasia may be harmless and occur on aparticular tissue. Hyperplasia may also be induced artificially.Hyperplasia may also occur abnormally, and is associated with a varietyof clinical diseases. Hyperplasia includes, but is not limited to, aneointimal hyperplasia of an artery; Keloids scars or hyperplasticscars; surgical adhesions; surgically induced hyperplasia; congenitaladrenal hyperplasia; endometrial hyperplasia; benign prostatichyperplasia; hyperplasia of the breast; focal epithelial hyperplasia;sebaceous hyperplasia; compensatory liver hyperplasia and such likehyperplastic conditions.

The term “subject” or “individual” or “patient” are used interchangeablyto refer to a member of an animal species of mammalian origin, includingbut not limited to, a mouse, a rat, a cat, a goat, sheep, horse,hamster, ferret, pig, a dog, a guinea pig, a rabbit and a primate, suchas, for example, a monkey, ape, or human.

The term “treat” or “treating” as used herein refers to accomplishingone or more of the following: (a) reducing the severity of a disorder;(b) limiting the development of symptoms characteristic of a disorderbeing treated; (c) limiting the worsening of symptoms characteristic ofa disorder being treated; (d) limiting the recurrence of a disorder inpatients that previously had the disorder; and (e) limiting recurrenceof symptoms in patients that were previously symptomatic for thedisorder. The term “disease” or “disorder” as used herein refers to animpairment of health or a condition of abnormal functioning. The term“syndrome” as used herein refers to a pattern of symptoms indicative ofsome disease or condition. The term “injury” as used herein refers todamage or harm to a structure or function of the body caused by anoutside agent or force, which may be physical or chemical. The term“condition” as used herein refers to a variety of health states and ismeant to include disorders or diseases caused by any underlyingmechanism or disorder, injury, and the promotion of healthy tissues andorgans. Disorders may include, for example, but not limited to,neoplasia or hyperplasia.

Compositions: Kinase Inhibiting Peptides (KIPs)

According to one aspect, the present invention provides a kinaseinhibiting composition, the composition comprising a therapeuticallyeffective amount of a kinase inhibiting peptide, wherein the kinaseinhibiting peptide inhibits kinase activity of a kinase enzyme.

According to one embodiment, the kinase inhibiting peptide is acyclin-dependent-kinase inhibitor. According to one such embodiment, thekinase inhibiting peptide is a peptide having the amino acid sequence ofthe general Formula IV:

Q1-Z1-Z2-Z3-Z4-Z5-Z6-Q2  [Formula IV]

wherein Q1 and Q2 are independently absent or present, and wherein if Q1and Q2 are present, Q1 and Q2 comprise a polypeptide of the sequence:

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11  [Formula IV(a)]

whereinX1 is any amino acid except K, or is absent;X2 is present or is absent; in some such embodiments when X2 is present,X2 is any amino acid; in some such embodiments, when X2 is present, X2is a hydrophobic amino acid; in some such embodiments, when X2 ispresent, X2 is A;X3 is any amino acid; in some such embodiments, X3 is a hydrophobicamino acid; in some such embodiments, X3 is selected from the groupconsisting of L, I, V, and M;X4 is any amino acid; in some such embodiments, X4 is selected from thegroup consisting of Q, A, and N;X5 is any basic amino acid;X6 is any amino acid; in some such embodiments, X6 is selected from thegroup consisting of Q and N;X7 is any amino acid; in some such embodiments, X7 is a hydrophobicamino acid; in some such embodiments, X7 is selected from the groupconsisting of L, I, V and M;X8 is any amino acid; in some such embodiments, X8 is selected from thegroup consisting of G, A, C, S, T and Y;X9 is any amino acid; in some such embodiments, X9 is a hydrophobicamino acid; in some such embodiments, X9 is selected from the groupconsisting of L, I, V and M; in some such embodiments, X9 is V;X10 is any amino acid;X11 is any amino acid, or is absent; andwherein Z1 and Z2 are present;wherein Z3 is present or absent, wherein Z4 is absent or present, but ifZ4 is present, Z3 is present, wherein Z5 is absent or present, but if Z5is present, Z3 and Z4 are present; wherein Z6 is absent or present, butif Z6 is present, Z3, Z4 and Z5 are present; andeach of Z1, Z2, Z3, Z4, Z5, and Z6, is a peptide selected from the groupconsisting of:

(a)X1-X2-B1-B2-X3-B3-X4  [Formula IV(b)],

wherein each of X1, X3 and X4 is a hydrophobic amino acid; X2 is anyamino acid; and, in some embodiments, X2 is a hydrophobic amino acid, Hor N; and wherein each of B1, B2 and B3 is a basic amino acid;

(b)X1-X2-B1-B2-X3-B3  [Formula IV(c)],

wherein each of X1 and X3 is a hydrophobic amino acid, X2 is any aminoacid, and, in some embodiments, X2 is a hydrophobic amino acid; H or N;and wherein each of B1, B2, and B3 is a basic amino acid;

(c)X1-X2-B1-B2-X3  [Formula IV(d)],

wherein each of X1 and X3 is a hydrophobic amino acid; X2 is any aminoacid; and, in some embodiments, X2 is a hydrophobic amino acid; H, or N;and wherein each of B1 and B2 is a basic amino acid;

(d)X1-B1-B2-X2-B3-X3  [Formula IV(e)],

wherein X1 is any amino acid; and, in some embodiments, X1 is ahydrophobic amino acid; H, or N; each of X2 and X3 is a hydrophobicamino acid, and each of B1, B2, and B3 is a basic amino acid;

(e)X1-B1-B2-X2-B3  [Formula IV(f)],

wherein X1 is a hydrophobic amino acid, H or N; X2 is any hydrophobicamino acid; and each of B1, B2, and B3 is a basic amino acid;

(f)X1-B1-B2-X2  [Formula IV(g)],

wherein X1 is any amino acid; and, in some embodiments, X1 is ahydrophobic amino acid, H or N; X2 is a hydrophobic amino acid; and eachof B1 and B2 is a basic amino acid;

(g)X1-X2-B1-B2  [Formula IV(h),

wherein X1 is a hydrophobic amino acid, X2 is any amino acid, and, insome embodiments, X2 is a hydrophobic amino acid, H or N; and each of B1and B2 is a basic amino acid;

(h)X1-X2-B1-X3-X4  [Formula IV(i)],

wherein each of X1, X3, and X4 is a hydrophobic amino acid; X2 is anyamino acid, and, in some embodiments, X2 is a hydrophobic amino acid, Hor N; and B1 is a basic amino acid;

(i)X1-B1-X2-X3  [Formula IV(j)],

wherein X1 is any amino acid, and, in some embodiments, X1 ishydrophobic amino acid, H or N; X2 is any amino acid, a hydrophobicamino acid or Q; X3 is a hydrophobic amino acid; and B1 is a basic aminoacid;

(j)X1-X2-B1-X3  [Formula IV(k)],

wherein each of X1 and X3 is a hydrophobic amino acid; X2 is any aminoacid, and, in some embodiments, X2 is a hydrophobic amino acid, H or N;and B1 is a basic amino acid;

(k)B1-B2-X1-X2-B3-X3  [Formula IV(1)],

wherein each of X1, X2, and X3 is a hydrophobic amino acid; and each ofB1, B2, and B3 is a basic amino acid;

(l)B1-B2-X1-X2-B3  [Formula IV(m)],

wherein each of X1 and X2 is a hydrophobic amino acid; and each of B1,B2, and B3 is a basic amino acid;

(m)B1-B2-X1-X2  [Formula IV(n)],

wherein each of X1 and X2 is a hydrophobic amino acid; and each of B1and B2 is a basic amino acid;

(n)B1-X1-X2-B2-B3-X3  [Formula IV(o)],

wherein each of X1, X2, and X3 is a hydrophobic amino acid; and each ofB1, B2, and B3 is a basic amino acid;

(o)B1-X1-X2-B2-B3  [Formula IV(p)],

wherein each of X1 and X2 is a hydrophobic amino acid, and each of B1,B2, and B3 is a basic amino acid;

(p)B1-X1-X2-B2  [Formula IV(q)],

wherein each of X1 and X2 is a hydrophobic amino acid; and each of B1and B2 is a basic amino acid;with the proviso that if Q2 is present, the two amino acids immediatelypreceding Q2 as part of Z2, Z3, Z4, Z5, or Z6 cannot be KA.

In another embodiment of the isolated polypeptide according to generalFormula IV, Q1, Z3, Z4, Z5, and Z6 are absent.

In some such embodiments, the kinase inhibiting peptide is a peptidehaving an amino acid sequence selected from the group consisting ofHRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]; WLRRIKAWLRRIKALARQLGVAA [SEQ IDNO: 113]; AND WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]. In some suchembodiments, the amino acid sequence of the kinase inhibiting peptide isHRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166]. In some such embodiments, theamino acid sequence of the kinase inhibiting peptide isWLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113]. In some such embodiments, theamino acid sequence of the kinase inhibiting peptide isWLRRIKAWLRRALARQLGVA [SEQ ID NO: 177].

According to one such embodiment, the kinase inhibiting peptide is KIP1.In some such embodiments, the KIP1 peptide is a peptide of at least 14amino acids, at least 21 amino acids, at least 28 amino acids, or amaximum of 35 amino acids. In some such embodiments, the KIP1 peptide isa peptide having an amino acid sequence according to Formula I:(XXBBXBX)_(n) wherein X is any amino acid, B is a basic amino acid, suchas, for example, K, R, H, and n is an integer between 2 and 5.

According to another such embodiment, the kinase inhibiting peptide isKIP2. In some such embodiments, the KIP2 peptide is a peptide of 11amino acids. In some such embodiments, the KIP2 peptide is a peptidehaving an amino acid sequence according to Formula II:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; wherein X1 is present or absent, andif present X1 is selected from the group consisting of A, F, I, L, V, W,and Y, or is an aromatic hydrophobic amino acid; X2 is any amino acid;X3 is selected from the group consisting of Q, N, R, and K; X4 isselected from the group consisting of A, G, I, L, V, R and K, or is analiphatic amino acid; X5 is selected from the group consisting of A, G,I, L, V, R and K, or is an aliphatic amino acid; X6 is selected from thegroup consisting of A, G, I, L, V, R and K, or is an aliphatic aminoacid; X7 is basic amino acid; X8 is selected from the group consistingof Q, N R, and K; X9 is selected from the group consisting of A, G, I,L, V, R and K, or is an aliphatic amino acid; X10 is present or absentprovided that if X10 is absent then X11 is also absent; if X10 ispresent, X10 is basic amino acid; and X11 is present or absent; if X11is present, X11 is selected from group consisting of A, G, I, L, V, Rand K, or is an aliphatic amino acid. In some such embodiments, X1 is F,W, or Y. In some such embodiments, X3 is R. In some such embodiments, X4is R or K. In some such embodiments, X5 is R or K. In some suchembodiments, X6 is R or K. In some such embodiments, X8 is Q or N. Insome such embodiments, X9 is R or K. In some such embodiments, X10 is Ror K. In some such embodiments, X11 is R or K.

According to another such embodiment, the kinase inhibiting peptide isKIP3. In some such embodiments, the KIP3 peptide is a peptide of atleast 12 amino acids, at least 18 amino acids, at least 24 amino acids,or at least 30 amino acids. The KIP3 amino acid sequence is of thegeneral Formula III: (XBBXBX)_(n) wherein X is any amino acid, B is abasic amino acid, such as, for example, K, R, H and n is an integerbetween 2 and 5.

According to another such embodiment, the kinase inhibiting peptide isKIP4. In some such embodiments, the KIP4 peptide is a peptide of atleast 16 amino acids, at least 24 amino acids, at least 32 amino acids,or at least 40 amino acids. In some such embodiments, the KIP4 peptideis a peptide having an amino acid sequence according to Formula V:(XXBBXBXX)_(n) wherein X is any amino acid, B is a basic amino acid,such as, for example, K, R, or H, and n is an integer between 2 and 5.

According to another embodiment, the kinase inhibiting peptide is apeptide having an amino acid sequence according to Formula VI:

Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2  [Formula VI],

wherein each of Z1 and Z2 is absent or is a transduction domain;X1 is absent or present, and if present is selected from the groupconsisting of A, KA, KKA, KKKA, and RA;X2 is an aliphatic amino acid or is selected from the group consistingof G, L, A, V, I, M, Y, W, and F;X3 is an aliphatic amino acid or is selected from the group consistingof V, L, I, A, G, Q, N, S, T, and C;X4 is selected from the group consisting of Q, N, H, R and K;X5 is selected from the group consisting of Q and N;X6 is an aliphatic amino acid or is selected from the group consistingof C, A, G, L, V, I, M, Y, W, and F;X7 is an aliphatic amino acid or is selected from the group consistingof S, A, C, T, and G;X8 is selected from the group consisting of V, L, I, and M;X9 is absent or is any amino acid; andX10 is absent or is any amino acid.

In some such embodiments, the kinase inhibiting peptide is a peptidehaving an amino acid sequence selected from the group consisting ofKAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173]; FAKLAARLYRKALARQLGVAA [SEQ IDNO: 163]; AND WLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142]. In some suchembodiments, the amino acid sequence of the kinase inhibiting peptide isKAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173]. In some such embodiments, theamino acid sequence of the kinase inhibiting peptide isFAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]. In some such embodiments, theamino acid sequence of the kinase inhibiting peptide isWLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142].

According to another embodiment, the kinase inhibiting peptide is akinase inhibiting peptide of Table 1.

TABLE 1 Additional KIP amino acid sequences Amino Acid Sequence[SEQ ID NO:]   1 (R)₄₋₉ [SEQ ID NO: 1]   2 GRKKRRQRRRPPQ [SEQ ID NO: 2]  3 AYARAAARQARA [SEQ ID NO: 3]   4 DAATATRGRSAASRPTERPRAPARSASRPRRPVE[SEQ ID NO: 4]   5 GWTLNSAGYLLGLINLKALAALAKKIL [SEQ ID NO: 5]   6PLSSIFSRIGDP [SEQ ID NO: 6]   7 AAVALLPAVLLALLAP [SEQ ID NO: 7]   8AAVLLPVLLAAP [SEQ ID NO: 8]   9 VTVLALGALAGVGVG [SEQ ID NO: 9]  10GALFLGWLGAAGSTMGAWSQP [SEQ ID NO: 10]  11 GWTLNSAGYLLGLINLKALAALAKKIL[SEQ ID NO: 11]  12 KLALKLALKALKAALKLA [SEQ ID NO: 12]  13KETWWETWWTEWSQPKKKRKV [SEQ ID NO: 13]  14 KAFAKLAARLYRKA [SEQ ID NO: 14] 15 KAFAKLAARLYRAA [SEQ ID NO: 15]  16 AAFAKLAARLYRKA [SEQ ID NO: 16] 17 KAFAKLAARLYRKA [SEQ ID NO: 17]  18 KAFAKLAARLYRKAGC [SEQ ID NO: 18] 19 KAFAKLAARLYRAAGC [SEQ ID NO: 19]  20 AAFAKLAARLYRKAGC[SEQ ID NO: 20]  21 KAFAKLAARLYRKAGC [SEQ ID NO: 21]  22KAFAKLAAQLYRKAGC [SEQ ID NO: 22]  23 AGGGGYGRKKRRQRRR [SEQ ID NO: 23] 24 (WLRRIKA)₁₋₃ [SEQ ID NO: 176]  25 YGRKKRRQRRR [SEQ ID NO: 24]  26YARAAARQARA [SEQ ID NO: 25]  27 RQRRKKRG [SEQ ID NO: 26]  28 GRKKRRQR[SEQ ID NO: 27]  29 YARAAARQARAKALNRQLGVA [SEQ ID NO: 28]  30YGRKKRRQRRRKALNRQLGVA [SEQ ID NO: 29]  31 GRKKRRQRKALNRQLGVA[SEQ ID NO: 30]  32 RQRRKKRGKALNRQLGVA [SEQ ID NO: 31]  33WLRRIKAWLRRIKAKALNRQLGVA [SEQ ID NO: 32]  34WLRRIKAWLRRIKAWLRRIKAKALNRQLGVA [SEQ ID NO: 33]  35YARAAARQARAKKKALNRQLGVA [SEQ ID NO: 34]  36 YGRKKRRQRRRKKKALNRQLGVA[SEQ ID NO: 35]  37 RQRRKKRGKKKALNRQLGVA [SEQ ID NO: 36]  38GRKKRRQRKKKALNRQLGVA [SEQ ID NO: 37]  39 WLRRIKAWLRRIKAKKKALNRQLGVA[SEQ ID NO: 38]  40 WLRRIKAWLRRIKAWLRRIKAKKKALNRQLGVA [SEQ ID NO: 39] 41 YARAAARQARAKKKALNRGLGVA [SEQ ID NO: 40]  42 YGRKKRRQRRRKKKALNRGLGVA[SEQ ID NO: 41]  43 RQRRKKRGKKKALNRGLGVA [SEQ ID NO: 42]  44GRKKRRQRKKKALNRGLGVA [SEQ ID NO: 43]  45 WLRRIKAWLRRIKAKKKALNRGLGVA[SEQ ID NO: 44]  46 WLRRIKAWLRRIKAWLRRIKAKKKALNRGLGVA [SEQ ID NO: 45] 47 YARAAARQARAKKKALNRQLAVA [SEQ ID NO: 46]  48 YGRKKRRQRRRKKKALNRQLAVA[SEQ ID NO: 47]  49 RQRRKKRGKKKALNRQLAVA [SEQ ID NO: 48]  50GRKKRRQRKKKALNRQLAVA [SEQ ID NO: 49]  51 WLRRIKAWLRRIKAKKKALNRQLAVA[SEQ ID NO: 50]  52 WLRRIKAWLRRIKAWLRRIKAKKKALNRQLAVA [SEQ ID NO: 51] 53 YARAAARQARAKKKALARQLGVA [SEQ ID NO: 52]  54 YGRKKRRQRRRKKKALARQLGVA[SEQ ID NO: 53]  55 RQRRKKRGKKKALARQLGVA [SEQ ID NO: 54]  56GRKKRRQRKKKALARQLGVA [SEQ ID NO: 55]  57 WLRRIKAWLRRIKAKKKALARQLGVA[SEQ ID NO: 56]  58 WLRRIKAWLRRIKAWLRRIKAKKKALARQLGVA [SEQ ID NO: 57] 59 YARAAARQARAKALNRGLGVA [SEQ ID NO: 58]  60 YGRKKRRQRRRKALNRGLGVA[SEQ ID NO: 59]  61 RQRRKKRGKALNRGLGVA [SEQ ID NO: 60]  62GRKKRRQRKALNRGLGVA [SEQ ID NO: 61]  63 WLRRIKAWLRRIKAKALNRGLGVA[SEQ ID NO: 62]  64 WLRRIKAWLRRIKAWLRRIKAKALNRGLGVA [SEQ ID NO: 63]  65YARAAARQARAKALNRQLAVA [SEQ ID NO: 64]  66 YGRKKRRQRRRKALNRQLAVA[SEQ ID NO: 65]  67 RQRRKKRGKALNRQLAVA [SEQ ID NO: 66]  68GRKKRRQRKALNRQLAVA [SEQ ID NO: 67]  69 WLRRIKAWLRRIKAKALNRQLAVA[SEQ ID NO: 68]  70 WLRRIKAWLRRIKAWLRRIKAKALNRQLAVA [SEQ ID NO: 69]  71YARAAARQARAKALARQLGVA [SEQ ID NO: 70]  72 YGRKKRRQRRRKALARQLGVA[SEQ ID NO: 71]  73 RQRRKKRGKALARQLGVA [SEQ ID NO: 72]  74GRKKRRQRKALARQLGVA [SEQ ID NO: 73]  75 WLRRIKAWLRRIKAKALARQLGVA[SEQ ID NO: 74]  76 WLRRIKAWLRRIKAWLRRIKAKALARQLGVA [SEQ ID NO: 75]  77YARAAARQARAKKKALNRGLGVAA [SEQ ID NO: 76]  78 YGRKKRRQRRRKKKALNRGLGVAA[SEQ ID NO: 77]  79 RQRRKKRGKKKALNRGLGVAA [SEQ ID NO: 78]  80GRKKRRQRKKKALNRGLGVAA [SEQ ID NO: 79]  81 WLRRIKAWLRRIKAKKKALNRGLGVAA[SEQ ID NO: 80]  82 WLRRIKAWLRRIKAWLRRIKAKKKALNRGLGVAA [SEQ ID NO: 81] 83 YARAAARQARAKKKALNRQLAVAA [SEQ ID NO: 82]  84YGRKKRRQRRRKKKALNRQLAVAA [SEQ ID NO: 83]  85 RQRRKKRGKKKALNRQLAVAA[SEQ ID NO: 84]  86 GRKKRRQRKKKALNRQLAVAA [SEQ ID NO: 85]  87WLRRIKAWLRRIKAKKKALNRQLAVAA [SEQ ID NO: 86]  88WLRRIKAWLRRIKAWLRRIKAKKKALNRQLAVAA [SEQ ID NO: 87]  89YARAAARQARAKKKALARQLGVAA [SEQ ID NO: 88]  90 YGRKKRRQRRRKKKALARQLGVAA[SEQ ID NO: 89]  91 RQRRKKRGKKKALARQLGVAA [SEQ ID NO: 90]  92GRKKRRQRKKKALARQLGVAA [SEQ ID NO: 91]  93 WLRRIKAWLRRIKAKKKALARQLGVAA[SEQ ID NO: 92]  94 WLRRIKAWLRRIKAWLRRIKAKKKALARQLGVAA [SEQ ID NO: 93] 95 YARAAARQARAKALNRGLGVAA [SEQ ID NO: 94]  96 YGRKKRRQRRRKALNRGLGVAA[SEQ ID NO: 95]  97 RQRRKKRGKALNRGLGVAA [SEQ ID NO: 96]  98GRKKRRQRKALNRGLGVAA [SEQ ID NO: 97]  99 WLRRIKAWLRRIKAKALNRGLGVAA[SEQ ID NO: 98] 100 WLRRIKAWLRRIKAWLRRIKAKALNRGLGVAA [SEQ ID NO: 99] 101YARAAARQARAKALNRQLAVAA [SEQ ID NO: 100] 102 YGRKKRRQRRRKALNRQLAVAA[SEQ ID NO: 101] 103 RQRRKKRGKALNRQLAVAA [SEQ ID NO: 102] 104GRKKRRQRKALNRQLAVAA [SEQ ID NO: 103] 105 WLRRIKAWLRRIKAKALNRQLAVAA[SEQ ID NO: 104] 106 WLRRIKAWLRRIKAWLRRIKAKALNRQLAVAA [SEQ ID NO: 105]107 YARAAARQARAKALARQLGVAA [SEQ ID NO: 106] 108 YGRKKRRQRRRKALARQLGVAA[SEQ ID NO: 107] 109 RQRRKKRGKALARQLGVAA [SEQ ID NO: 108] 110GRKKRRQRKALARQLGVAA [SEQ ID NO: 109] 111 WLRRIKAWLRRIKAKALARQLGVAA[SEQ ID NO: 110] 112 WLRRIKAWLRRIKAWLRRIKAKALARQLGVAA [SEQ ID NO: 111]113 WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113] 114 WLRRIKAWLRRIKALNRQLGVAA[SEQ ID NO: 142] 115 FAKLAARLYRKA [SEQ ID NO: 160] 116 FAKLAARLYRKAGC[SEQ ID NO: 161] 117 FAKLAARLYRKALNRQLGVAA [SEQ ID NO: 162] 118FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163] 119 FAKLAARLYRKALNRQLGVA[SEQ ID NO: 164] 120 FAKLAARLYRKALARQLGVA [SEQ ID NO: 165] 121HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166] 122 WLRRIKAHRRIKALARQLGVAA[SEQ ID NO: 167] 123 WLRRIKAWLRR [SEQ ID NO: 168] 124WLRRIKAWLRRALNRQLGVAA [SEQ ID NO: 169] 125 YARAAARQARAKALNRQLGVAA[SEQ ID NO: 147] 126 YARAAARQARALNRQLGVAA [SEQ ID NO: 170] 127YARAAARQARALARQLGVAA [SEQ ID NO: 171] 128 KAFAKLAARLYRKALNRQLGVAA[SEQ ID NO: 172] 129 KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173] 130KAFAKLAARLYRKALNRQLGVA [SEQ ID NO: 174] 131 KAFAKLAARLYRKALARQLGVA[SEQ ID NO: 175] 132 WLRRIKAWLRRALARQLGVA [SEQ ID NO: 177]

The following terms are used herein to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

The term “reference sequence” refers to a sequence used as a basis forsequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

The term “comparison window” refers to a contiguous and specifiedsegment of a polynucleotide sequence, wherein the polynucleotidesequence may be compared to a reference sequence and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be at least 30contiguous nucleotides in length, at least 40 contiguous nucleotides inlength, at least 50 contiguous nucleotides in length, at least 100contiguous nucleotides in length, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence, a gap penaltytypically is introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson, et al., Methods in Molecular Biology 24:307-331(1994). The BLAST family of programs, which can be used for databasesimilarity searches, includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters. Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center forBiotechnology-Information (http://www.hcbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits then are extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a word length (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins may be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats, or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs may be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17:191-201 (1993)) low-complexity filters may beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window. When percentage of sequence identityis used in reference to proteins it is recognized that residue positionsthat are not identical often differ by conservative amino acidsubstitutions, i.e., where amino acid residues are substituted for otheramino acid residues with similar chemical properties (e.g. charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, at least 80% sequence identity, at least 90% sequence identityand at least 95% sequence identity, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill will recognize that these values may be adjustedappropriately to determine corresponding identity of proteins encoded bytwo nucleotide sequences by taking into account codon degeneracy, aminoacid similarity, reading frame positioning and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 60%, or at least 70%, at least 80%, atleast 90%, or at least 95%. Another indication that nucleotide sequencesare substantially identical is if two molecules hybridize to each otherunder stringent conditions. However, nucleic acids that do not hybridizeto each other under stringent conditions are still substantiallyidentical if the polypeptides that they encode are substantiallyidentical. This may occur, e.g., when a copy of a nucleic acid iscreated using the maximum codon degeneracy permitted by the geneticcode. One indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide that the first nucleic acid encodes isimmunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, at least 80%, at least 85%, at least 90% or 95%sequence identity to the reference sequence over a specified comparisonwindow. Optionally, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443(1970). An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides which are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

According to another embodiment, the present invention provides anisolated nucleic acid that encodes a polypeptide having at least 85%amino acid sequence identity to KIP1, wherein the polypeptide inhibitskinase activity of a kinase enzyme. In some such embodiments, theisolated nucleic acid encodes a polypeptide with 100% amino acidsequence identity to KIP1, wherein the polypeptide inhibits kinaseactivity of a kinase enzyme. In some such embodiments, the KIP1 sequenceis operably linked to a controllable regulatory element.

According to another embodiment, the present invention provides anisolated nucleic acid that encodes a polypeptide having at least 85%amino acid sequence identity to KIP2, wherein the polypeptide inhibitskinase activity of a kinase enzyme. In some such embodiments, theisolated nucleic acid encodes a polypeptide with 100% amino acidsequence identity to KIP2, wherein the polypeptide inhibits kinaseactivity of a kinase enzyme. In some such embodiments, the KIP2 sequenceis operably linked to a controllable regulatory element.

According to another embodiment, the present invention provides anisolated nucleic acid that encodes a polypeptide having at least 85%amino acid sequence identity to KIP3, wherein the polypeptide inhibitskinase activity of a kinase enzyme. In some such embodiments, theisolated nucleic acid encodes a polypeptide with 100% amino acidsequence identity to KIP3, wherein the polypeptide inhibits kinaseactivity of a kinase enzyme. In some such embodiments, the KIP3 sequenceis operably linked to a controllable regulatory element.

According to another embodiment, the present invention provides anisolated nucleic acid that encodes a polypeptide having at least 85%amino acid sequence identity to KIP4, wherein the polypeptide inhibitskinase activity of a kinase enzyme. In some such embodiments, theisolated nucleic acid encodes a polypeptide with 100% amino acidsequence identity to KIP4, wherein the polypeptide inhibits kinaseactivity of a kinase enzyme. In some such embodiments, the KIP4 sequenceis operably linked to a controllable regulatory element.

According to another embodiment, the present invention provides anisolated nucleic acid that specifically hybridizes to mRNA encoding apeptide comprising a KIP1 or KIP2 amino acid sequence. The term“specifically hybridizes” as used herein refers to the process of anucleic acid distinctively or definitively forming base pairs withcomplementary regions of at least one strand of DNA that was notoriginally paired to the nucleic acid. For example, a nucleic acid thatmay bind or hybridize to at least a portion of an mRNA of a cellencoding a peptide comprising a KIP sequence may be considered a nucleicacid that specifically hybridizes. A nucleic acid that selectivelyhybridizes undergoes hybridization, under stringent hybridizationconditions, of the nucleic acid sequence to a specified nucleic acidtarget sequence to a detectably greater degree (e.g., at least 2-foldover background) than its hybridization to non-target nucleic acidsequences and to the substantial exclusion of non-target nucleic acids.Selectively hybridizing sequences typically have about at least 80%sequence identity, at least 90% sequence identity, or at least 100%sequence identity (i.e., complementary) with each other.

Methods of extraction of RNA are well-known in the art and aredescribed, for example, in J. Sambrook et al., “Molecular Cloning: ALaboratory Manual” (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989), vol. 1, ch. 7, “Extraction, Purification, andAnalysis of Messenger RNA from Eukaryotic Cells,” incorporated herein bythis reference. Other isolation and extraction methods are alsowell-known, for example in F. Ausubel et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, 2007). Typically, isolation isperformed in the presence of chaotropic agents, such as guanidiniumchloride or guanidinium thiocyanate, although other detergents andextraction agents alternatively may be used. Typically, the mRNA isisolated from the total extracted RNA by chromatography overoligo(dT)-cellulose or other chromatographic media that have thecapacity to bind the polyadenylated 3′-portion of mRNA molecules.Alternatively, but less preferably, total RNA can be used. However, itis generally preferred to isolate poly(A)+RNA from mammalian sources.

According to another embodiment, the present invention provides anantibody or an antibody fragment that specifically binds to an aminoacid sequence of a KIP peptide.

Methods of Inhibiting Kinase Activity

According to another aspect, the present invention provides a method forinhibiting kinase activity of a kinase enzyme, the method comprising thesteps: (a) providing a kinase inhibiting composition, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide; (b) contacting the kinase inhibiting compositionwith a kinase enzyme such that the kinase inhibiting peptide associateswith the kinase enzyme; and (c) reducing the kinase activity of thekinase enzyme.

According to another embodiment, the present invention provides a methodfor inhibiting kinase activity of a kinase enzyme, the method comprisingthe steps of: (a) providing a kinase inhibiting composition, wherein thekinase inhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide furthercomprises a KIP1 peptide; (b) contacting the kinase inhibitingcomposition with a kinase enzyme such that the kinase inhibiting peptideassociates with kinase enzyme; and (c) reducing the kinase activity ofthe kinase enzyme.

According to another embodiment, the present invention provides a methodfor inhibiting kinase activity of a kinase enzyme, the method comprisingthe steps of: (a) providing a kinase inhibiting composition, wherein thekinase inhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide furthercomprises a KIP2 peptide; (b) contacting the kinase inhibitingcomposition with a kinase enzyme such that the kinase inhibiting peptideassociates with the kinase enzyme; and (c) reducing the kinase activityof the kinase enzyme.

According to another embodiment, the present invention provides a methodfor inhibiting kinase activity of a kinase enzyme, the method comprisingthe steps of: (a) providing a kinase inhibiting composition, wherein thekinase inhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide furthercomprises a KIP3 peptide; (b) contacting the kinase inhibitingcomposition with a kinase enzyme such that the kinase inhibiting peptideassociates with the kinase enzyme; and (c) reducing the kinase activityof the kinase enzyme.

According to another embodiment, the present invention provides a methodfor lowering the enzymatic velocity of a kinase reaction, the methodcomprising the steps: (a) providing a kinase inhibiting composition,wherein the kinase inhibiting composition comprises an inhibitory amountof a kinase inhibiting peptide, wherein the kinase inhibiting peptidecomprises a KIP1 peptide, (b) contacting the kinase inhibitingcomposition with a kinase enzyme such that the kinase inhibiting peptideassociates with the kinase enzyme; and (c) lowering the enzymaticvelocity of the kinase enzyme. In some such embodiments, the kinaseenzyme is in a prokaryotic cell. In some such embodiments, the kinaseenzyme is in a eukaryotic cell. In some such embodiments, the kinaseinhibiting peptide is operably linked to a controllable regulatoryelement.

According to another embodiment, the present invention provides a methodfor lowering the enzymatic velocity of a kinase reaction, the methodcomprising the steps: (a) providing a kinase inhibiting composition,wherein the kinase inhibiting composition comprises an inhibitory amountof a kinase inhibiting peptide, wherein the kinase inhibiting peptidecomprises a KIP2 peptide, (b) contacting the kinase inhibitingcomposition with the kinase enzyme such that the kinase inhibitingpeptide associates with the kinase enzyme; and (c) lowering theenzymatic velocity of the kinase enzyme. In some such embodiments, thekinase enzyme is in a prokaryotic cell. In some such embodiments, thekinase enzyme is in a eukaryotic cell. In some such embodiments, thekinase inhibiting peptide is operably linked to a controllableregulatory element.

According to another embodiment, the kinase enzyme is a serine kinase.According to another embodiment, the kinase enzyme is a threoninekinase. According to another embodiment, the kinase enzyme is a tyrosinekinase. According to another embodiment, the kinase enzyme is a receptortyrosine kinase. According to another embodiment, the kinase enzyme is aserine/threonine kinase.

According to another embodiment, the kinase enzyme is a kinase enzymeselected from the group consisting of: Ab 1, Akt/PKB, AMPK, Arg, Ask,Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI, CaMKIδ, CaMKIIβ, CaMKIIγ,CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin, CK1y1, CK1y2, CK1y3, Ck1δ,CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2 mutants, CK1δ, CK2, c-Kit,CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR, DYRK2, EGFR, Ephs, EphA2,FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit, Flt3, Flt4, Fms/CSF-1 R, Fyn,GRK5, GRK6, GRK7, GSK, CSK3, Hck, HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1,ICF IR, IKK, Insulin R, IRAK, IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3,JNK/SAPK, KDR, Lck, LIMK, LIMK1, LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase,MEK, MEK1, MELK, Met, Mer, MINK, MKK, MLCK, MLK1, MRCKa, MSK1, MST,MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα, PDGFRβ, PDK, PhKγ2, PI 3-Kinase,PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1, PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI,PKCδ, PKG1, PKG1α, PKG1β, PKR, PLK, PRAK, PTK5, Pyk, Raf, Rct, RIPK2,ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAP Kinase, S6 Kinase,Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src, Syk, TAK1, TAO1, TAO2, TBK,Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3, Ulk2, VRK2, WEE, Yes,ZAP-70 and ZIPK.

In some such embodiments, the kinase enzyme is selected from the groupconsisting of a ROCK kinase, a Src kinase, a PKC kinase and a Trkkinase. In some such embodiments, the kinase enzyme is a ROCK kinase. Insome such embodiments, the kinase enzyme is ROCK-1. In some suchembodiments, the kinase enzyme is a Src kinase. In some suchembodiments, the kinase enzyme is SRrc(1-530). In some such embodiments,the kinase enzyme is a PKC kinase. In some such embodiments, the kinaseenzyme is PKCβ1 or PKCδ. In some such embodiments, the kinase enzyme isa Trk kinase. In some such embodiments, the kinase enzyme is TrkA.

According to another embodiment, the present invention provides a methodof inhibiting mRNA translation of an mRNA_(KIP) molecule, the methodcomprising the steps: (a) providing a kinase inhibiting composition,wherein the kinase inhibiting composition comprises an inhibitory amountof an isolated nucleic acid that encodes a polypeptide having 100% aminoacid sequence identity to KIP1, wherein the polypeptide inhibits kinaseactivity of a kinase enzyme; (b) contacting the kinase inhibitingcomposition of step (a) with an mRNA_(KIP) molecule such that theisolated nucleic acid hybridizes with the mRNA_(KIP); and (c) inhibitingthe mRNA translation of the mRNA_(KIP) molecule. As used herein, theterm “mRNA_(KIP)” refers to an mRNA molecule that upon mRNA translation,yields a KIP molecule. In some such embodiments, the mRNA translation ofthe mRNA_(KIP) molecule yields a KIP1 molecule. In some suchembodiments, the mRNA_(KIP) molecule comprises a KIP sequence operablylinked to a controllable regulatory element. In some such embodiments,in step (a) the isolated nucleic acid encodes a polypeptide having about85% amino acid sequence identity to a KIP1 peptide.

According to another embodiment, the present invention provides a methodof inhibiting mRNA translation of an mRNA_(KIP) molecule, the methodcomprising the steps: (a) providing a kinase inhibiting composition,wherein the kinase inhibiting composition comprises an inhibitory amountof an isolated nucleic acid that encodes a polypeptide having 100% aminoacid sequence identity to a KIP2 peptide, wherein the polypeptideinhibits kinase activity of a kinase enzyme; (b) contacting the kinaseinhibiting composition of step (a) with an mRNA_(KIP) molecule such thatthe isolated nucleic acid hybridizes with the mRNA_(KIP); and (c)inhibiting the mRNA translation of the mRNA_(KIP) molecule. In some suchembodiments, the mRNA translation of the mRNA_(KIP) molecule yields aKIP2 molecule. In some such embodiments, the mRNA_(KIP) moleculecomprises a KIP sequence operably linked to a controllable regulatoryelement. In some such embodiments, in step (a) the isolated nucleic acidencodes a polypeptide having about 85% amino acid sequence identity to aKIP2 peptide.

According to another embodiment, the present invention provides a methodof inhibiting a kinase inhibitory function of a KIP1 peptide, the methodcomprising the steps: (a) providing a kinase inhibiting composition,wherein the kinase inhibiting composition comprises a therapeuticallyeffective amount of an antibody specific to an epitope of a kinaseinhibiting peptide amino acid sequence, wherein the kinase inhibitingpeptide amino acid sequence is a KIP1 peptide amino acid sequence; (b)contacting the kinase inhibiting composition of step (a) with a KIP1peptide such that the antibody associates with the KIP1 peptide; and (c)inhibiting the kinase inhibitory function of the kinase inhibitingpeptide.

According to another embodiment, the present invention provides a methodof inhibiting the kinase inhibitory function of a KIP2 peptide, themethod comprising the steps: (a) providing a kinase inhibitingcomposition, wherein the kinase inhibiting composition comprises atherapeutically effective amount of an antibody specific to an epitopeof a kinase inhibiting peptide amino acid sequence, wherein the kinaseinhibiting peptide amino acid sequence is a KIP2 peptide amino acidsequence; (b) contacting kinase inhibiting composition of step (a) witha KIP2 peptide such that the antibody associates with the KIP2 peptide;and (c) inhibiting the kinase inhibitory function of the kinaseinhibiting peptide.

Inhibiting Disorders

According to another aspect, the present invention provides a method forinhibiting hyperplasia of a cell population, the method comprising thesteps: (a) providing a therapeutically effective amount of a kinaseinhibiting composition to a subject in need thereof, wherein the kinaseinhibiting composition comprises an isolated nucleic acid that encodes apolypeptide having 100% amino acid sequence identity to a KIP peptide,wherein the polypeptide inhibits kinase activity of a kinase enzyme; (b)contacting the kinase inhibiting composition of step (a) with at leastone hyperplastic cell such that the isolated nucleic acid associateswith the at least one hyperplastic cell; and (c) inhibiting thehyperplasia. In another embodiment, the isolated nucleic acid of step(a) further comprises a controllable regulatory element.

According to another aspect, the present invention provides a method forinhibiting hyperplasia of a cell population, the method comprising thesteps: (a) providing a therapeutically effective amount of a kinaseinhibiting composition to a subject in need thereof, wherein the kinaseinhibiting composition comprises an inhibiting amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide comprises aKIP peptide, (b) contacting the kinase inhibitory composition with atleast one hyperplastic cell such that the kinase inhibiting peptideassociates with the at least one hyperplastic cell; and (c) inhibitingthe hyperplasia. In some such embodiments, the kinase inhibiting peptideof step (a) is operably linked to a controllable regulatory element.

The term “growth” as used herein refers to a process of becoming larger,longer or more numerous, or an increase in size, number, or volume ofcells in a cell population.

According to another aspect, the present invention provides a method forinhibiting growth of a neoplasm, the method comprising the steps: (a)providing a therapeutically effective amount of a kinase inhibitingcomposition to a subject in need thereof, wherein the kinase inhibitingcomposition comprises an inhibitory amount of an isolated nucleic acidthat encodes a polypeptide having 100% amino acid sequence identity to aKIP peptide, wherein the polypeptide inhibits kinase activity of akinase enzyme; (b) contacting the neoplasm with the kinase inhibitingcomposition of step (a) such that the isolated nucleic acid associateswith the neoplasm; and (c) inhibiting the growth of the neoplasm. Inanother embodiment, the isolated nucleic acid of step (a) furthercomprises a controllable regulatory element.

According to another aspect, the present invention provides a method forinhibiting growth of a neoplasm, the method comprising the steps: (a)providing a therapeutically effective amount of a kinase inhibitingcomposition to a subject in need thereof, wherein the kinase inhibitingcomposition comprises an inhibitory amount of a kinase inhibitingpeptide, wherein the kinase inhibiting peptide comprises a KIP peptide,(b) contacting the neoplasm with the kinase inhibiting composition suchthat the kinase inhibiting peptide associates with the neoplasm; and (c)inhibiting the growth of the neoplasm. In some such embodiments, thekinase inhibiting peptide of step (a) is operably linked to acontrollable regulatory element.

According to another embodiment, the neoplasm is a benign tumor.According to another embodiment, the neoplasm is a malignant tumor.According to another embodiment, the neoplasm is a neoplasm selectedfrom the group consisting of: a papilloma, an adenoma, a hydatidiformmole, a fibroma, a chondroma, an osteoma, a leiomyoma, a rhabdomyoma, alipoma, a hemangioma, a lymphangioma, a polycythemia vera, an infectiousmononucleosis, a “benign” glioma, a meningioma, a ganglioneuroma, aneurilemmoma, a neurofibroma, a pigmented nevus (mole), apheochromocytoma, a carcinoid tumors, a teratoma, a carcinoma, anadenocarcinoma, a basal cell carcinoma, a choriocarcinoma, afibrosarcoma, a chondrosarcoma, an osteosarcoma, a leiomyosarcoma, arhabdomyosarcoma, a liposarcoma, a hemangiosarcoma, a lymphangiosarcoma,a myelocytic leukemia, an erythrocytic leukemia, a lymphocytic leukemia,a multiple myeloma, a monocytic leukemia, an Ewing's sarcoma, anon-Hodgkin's malignant lymphoma, a medulloblastoma, anoligodendroglioma, a neurilemmal sarcoma malignant melanoma, thymoma, aglioblastoma multiforme, an astrocytoma, an ependymoma, an meningealsarcoma, a neuroblastoma (schwannoma), a neurofibrosarcoma, a malignantpheochromocytoma, a retinoblastoma, a carcinoid tumor, a nephroblastoma(Wilms' tumor), a teratocarcinoma and an embryonal carcinoma withchoriocarcinoma.

According to another embodiment, the isolated nucleic acid that encodesa polypeptide having 100% amino acid sequence identity to a KIP peptide,wherein the polypeptide inhibits kinase activity, may be administeredlocally or systemically.

According to another aspect, the present invention further describesexperiments in animal models of human disease that will be used todetermine the effect of the polypeptides of the present invention. Theseanimal models have been used by other investigators, and are generallyaccepted as such. The therapeutic results obtained with this modeltherefore can be extrapolated to methods of treating human subjects.

Model of Hyperplasia

A KIP peptide may be evaluated for its ability to reduce hyperplasiausing a porcine model of coronary restenosis as described by Heldman etal. (A. W. Heldman, L. Cheng, G. M. Jenkins, etc., 2001, “PaclitaxelStent Coating Inhibits Neointimal Hyperplasia at 4 Weeks in a PorcineModel of Coronary Restenosis,” Circulation, 103: 2289-2295). Stents arecoated by dipping the stent into a solution containing a KIP peptide anddrying the solvent. The coated stents is mounted on balloon cathetersand sterilized using ethylene oxide gas. Male and female NIH minipigsweighing 35 to 45 kg are pretreated with aspirin (325 mg) and diltiazem(180 mg) the day before stent implantation. After sedating the animalswith ketamine (20 mg/kg IM) and acetylpromazine (0.22 mg/kg IM) andgiving the animas sodium pentobarbital (4 mg/kg IV) to facilitate supinepositioning and endotracheal intubation, an arterial sheath is insertedinto the right carotid artery under sterile surgical technique. Afteradministering heparin (5000 U), the stent may be delivered to the leftanterior descending coronary artery through a guiding catheter anddeployed using balloon inflations. Angiograms are performed throughoutthe recovery and experimental period. The extent of hyperplasia may beevaluated inter alia using histological preparation andhistomorphometric analysis.

Model of Neoplasm

A KIP peptide may be evaluated for its ability of a KIP peptide toinhibit or decrease the growth of a neoplasm using a scid mouse tumormodel, such as that described by Becker et al. (J. C. Becker, N. Varki,S. D. Gillies, K. Furukawa, and R. A. Reisfeld, 1996, “Long-lived andTransferable Tumor Immunity in Mice after Targeted Interleukin-2Therapy,” J. Clin. Invest., 98(12): 2801-2804). Subcutaneous tumors maybe induced by subcutaneous injection of 5×10⁶ tumor cells (such as themurine melanoma cell line B16 or the cell line B78-D14) suspended in anisotonic buffer, such as RPMI 1640. Within 14 days, tumors of volumearound 40 μl should be present. The KIP peptide may be injected directlyinto the tumor mass, administered at the time of tumor induction viainjection, or delivered via a vehicle or a device. The subcutaneoustumors may be evaluated, inter allia, via histology,immunohistochemistry, or imaging techniques to evaluate the efficacy ofthe KIP peptide in inhibiting, slowing, decreasing, or modifying thegrowth or size of the neoplasm.

Cell Death

The term “programmed cell death” (or “PCD”) as used herein refers todeath of a cell in any form, mediated by an intracellular program. Incontrast to necrosis (a form of cell-death that results from acutetissue injury and provokes an inflammatory response), PCD is a regulatedprocess that generally confers advantage during the life cycle of anorganism. Two types of PCD are known: apotosis (Type I) and autophagic(Type II) cell death. Other pathways of cell death include non-apoptoticprogrammed cell death (also referred to as caspase-independent PCD ornecrosis-like PCD), anolkis (a form of apoptosis induced byanchorage-dependent cells detaching from the surrounding extracellularmatrix), cornification, excitotoxicity, and Wallerain degeneration(axonal degeneration).

The term “apoptosis” as used herein refers to a series of biochemicalevents that lead to morphological changes including, for example, butnot limited to, blebbing, changes to the cell membrane, such as loss ofmembrane asymmetry and attachment, cell shrinkage, nuclearfragmentation, chromatin condensation, and chromosomal DNAfragmentation. The process of apoptosis is controlled by a diverse rangeof extracellular and intracellular cell signals. Such extracellularsignals may include, for example, but are not limited to, toxins,hormones, growth factors, nitric oxide or cytokines, and therefore musteither cross the plasma membrane or transduce the cell to effect aresponse. The term “intracellular apoptotic signaling” as used hereinrefers to a response initiated by a cell in response to stress. Itultimately may result in cell suicide. The binding of nuclear receptorsby factors, such as, glucocorticoids, heat, radiation, nutrientdeprivation, viral infection, and hypoxia may lead to the release ofintracellular apoptotic signals by a damaged cell.

According to another aspect, the present invention provides a method forinducing programmed cell death in a cell population, the methodcomprising the steps: (a) providing a therapeutically effective amountof a kinase inhibiting composition, wherein the kinase inhibitingcomposition comprises an inhibitory amount of a kinase inhibitingpeptide; (b) contacting at least one cell in the cell population withthe kinase inhibiting composition such that the kinase inhibitingpeptide associates with the at least one cell; and (c) inducingprogrammed cell death of the at least one cell in the cell population.In some such embodiments, the cell is a prokaryotic cell. In some suchembodiments, the cell is a eukaryotic cell. In some such embodiments,the cell is a mammalian cell. In some such embodiments, the mammaliancell is selected from the group consisting of a neuron, an epithelialcell, a muscle cell, a blood cell, an immune cell, a stem cell, anosteocyte, or an endothelial cell. In some such embodiments, theprogrammed cell death is apoptosis.

According to another embodiment, the present invention provides a methodfor inducing programmed cell death in a cell population, the methodcomprising the steps: (a) providing a therapeutically effective amountof a kinase inhibiting composition, wherein the kinase inhibitingcomposition comprises an inhibitory amount of an isolated nucleic acidthat encodes a polypeptide having 100% amino acid sequence identity to aKIP peptide, wherein the polypeptide inhibits kinase activity of akinase enzyme; (b) contacting at least one cell in the cell populationwith the kinase inhibiting composition of step (a) such that theisolated nucleic acid associates with the at least one cell; and (c)inducing programmed cell death of the at least one cell in the cellpopulation. In some such embodiments, the isolated nucleic acid of step(a) further comprises a controllable regulatory element. In some suchembodiments, the cell is a prokaryotic cell. In some such embodiments,the cell is a mammalian cell. In some such embodiments, the mammaliancell is selected from the group consisting of a neuron, an epithelialcell, a muscle cell, a blood cell, an immune cell, a stem cell, anosteocyte, or an endothelial cell. In some such embodiments, the cell isa eukaryotic cell. In some such embodiments, the programmed cell deathis apoptosis.

The term “progressive disease” as used herein implies that thepreviously administered treatment(s) are/were not effective and othertreatments may be necessary to control the disease. The term “stabledisease” as used herein refers to no significant decrease in the size ornumber of lesions in the body, and implies further treatment(s) probablywill be needed to attempt a cure. The term “partial response” as usedherein refers to a reduction of disease by about 30% or more asidentified in clinical examination, X-ray, scans, or tests forbiomarkers. The term “complete response” as used herein refers to noresidual disease that can be identified in clinical examination, byX-ray, or in tests for biomarkers of the disease; cure is not implied.

According to another aspect, the present invention provides a method forinhibiting the progression of a proliferating cell population, themethod comprising the steps of: (a) providing a therapeuticallyeffective amount of a kinase inhibiting composition, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide (b) contacting at least one cell in the proliferatingcell population with the kinase inhibiting composition such that thekinase inhibiting peptide associates with the proliferating at least onecell; and (c) inhibiting proliferation by the at least one cell. In somesuch embodiments, the kinase inhibiting peptide is operably linked to acontrollable regulatory element. In some such embodiments, the cell is aprokaryotic cell. In some such embodiments, the cell is a eukaryotic.

According to one embodiment, the present invention provides a method forinhibiting the progression of a proliferating cell population, themethod comprising the steps of: (a) providing a therapeuticallyeffective amount of a kinase inhibiting composition, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide comprises aKIP peptide, (b) contacting at least one cell in the proliferating cellpopulation with the kinase inhibiting composition such that the kinaseinhibiting peptide associates with the proliferating at least one cell;and (c) inhibiting proliferation by the at least one cell. In some suchembodiments, the cell is a prokaryotic cell. In some such embodiments,the cell is a eukaryotic cell.

According to another embodiment, the present invention provides a methodfor inhibiting the progression of a proliferating cell population, themethod comprising the steps: (a) providing a therapeutically effectiveamount of a kinase inhibiting composition, wherein the kinase inhibitingcomposition comprises an inhibitory amount of an isolated nucleic acidthat encodes a polypeptide having 100% amino acid sequence identity to aKIP peptide, wherein the polypeptide inhibits kinase activity of akinase enzyme; (b) contacting at least one cell in the proliferatingcell population with the kinase inhibiting composition of step (a) suchthat the isolated nucleic acid associates with the proliferating atleast one cell; and (c) inhibiting proliferation of the at least onecell. In another embodiment, the isolated nucleic acid of step (a)further comprises a controllable regulatory element. In some suchembodiments, the cell is a prokaryotic cell. In some such embodiments,the cell is a eukaryotic cell.

According to another embodiment, the kinase inhibiting composition,wherein it is desirable to deliver them locally, may be formulated forparenteral administration by injection, e.g., by bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The pharmaceutical compositions (i.e., kinase inhibiting compositions)also may comprise suitable solid or gel phase carriers or excipients.Examples of such carriers or excipients include, but are not limited to,calcium carbonate, calcium phosphate, various sugars, starches,cellulose derivatives, gelatin, and polymers such as polyethyleneglycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, microencapsulated, and if appropriate, with one or moreexcipients, encochleated, coated onto microscopic gold particles,contained in liposomes, pellets for implantation into the tissue, ordried onto an object to be rubbed into the tissue. Such pharmaceuticalcompositions also may be in the form of granules, beads, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, drops or preparations with protractedrelease of active compounds, in whose preparation excipients andadditives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, or solubilizers are customarilyused as described above. The pharmaceutical compositions are suitablefor use in a variety of drug delivery systems. For a brief review ofmethods for drug delivery, see Langer 1990 Science 249, 1527-1533, whichis incorporated herein by reference.

The kinase inhibiting composition, and optionally other therapeutics,may be administered per se (neat) or in the form of a pharmaceuticallyacceptable salt. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts may beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group. By“pharmaceutically acceptable salt” is meant those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell-known in the art. For example, P. H. Stahl, et al. describepharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002). The salts may be prepared in situ during thefinal isolation and purification of the compounds described within thepresent invention or separately by reacting a free base function with asuitable organic acid. Representative acid addition salts include, butare not limited to, acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsmay be also obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

The formulations may be presented conveniently in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association a kinaseinhibiting composition, or a pharmaceutically acceptable salt or solvatethereof (“active compound”) with the carrier which constitutes one ormore accessory agents. In general, the formulations are prepared byuniformly and intimately bringing into association the active agent withliquid carriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

The pharmaceutical agent or a pharmaceutically acceptable ester, salt,solvate or prodrug thereof may be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action. Solutions or suspensions used for parenteral,intradermal, subcutaneous, intrathecal, or topical application mayinclude, but are not limited to, for example, the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Administered intravenously, particularcarriers are physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants including preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release may be controlled.Such long acting formulations may be formulated with suitable polymericor hydrophobic materials (for example as an emulsion in an acceptableoil) or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissues.

The locally injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions may be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral (including but not limited to, subcutaneous,intradermal, intramuscular, intravenous, intrathecal and intraarticular)administration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes, which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline, water-for-injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Another method of formulation of the compositions described hereininvolves conjugating the compounds described herein to a polymer thatenhances aqueous solubility. Examples of suitable polymers include butare not limited to polyethylene glycol, poly-(d-glutamic acid),poly-(l-glutamic acid), poly-(l-glutamic acid), poly-(d-aspartic acid),poly-(l-aspartic acid), poly-(l-aspartic acid) and copolymers thereof.Polyglutamic acids having molecular weights between about 5,000 to about100,000, with molecular weights between about 20,000 and about 80,000may be used and with molecular weights between about 30,000 and about60,000 may also be used. The polymer is conjugated via an ester linkageto one or more hydroxyls of an inventive epothilone using a protocol asessentially described by U.S. Pat. No. 5,977,163 which is incorporatedherein by reference. Particular conjugation sites include the hydroxyloff carbon-21 in the case of 21-hydroxy-derivatives of the presentinvention. Other conjugation sites include, but are not limited, to thehydroxyl off carbon 3 and/or the hydroxyl off carbon 7.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

In some embodiments, the kinase inhibiting composition is apharmaceutical composition. The pharmaceutical compositions describedwithin the present invention contain a therapeutically effective amountof a kinase inhibiting composition and optionally other therapeuticagents included in a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein refers to one ormore compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration to a human or othervertebrate animal. The term “carrier” as used herein refers to anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. The activeingredient may be a kinase inhibiting composition. The components of thepharmaceutical compositions also are capable of being commingled in amanner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficiency.

The therapeutic agent(s), including the kinase inhibiting composition,may be provided in particles. The term “particles” as used herein refersto nano or microparticles (or in some instances larger) that may containin whole or in part the kinase inhibiting composition. The particles maycontain the therapeutic agent(s) in a core surrounded by a coating. Thetherapeutic agent(s) also may be dispersed throughout the particles. Thetherapeutic agent(s) also may be adsorbed on at least one surface of theparticles. The particles may be of any order release kinetics, includingzero order release, first order release, second order release, delayedrelease, sustained release, immediate release, etc., and any combinationthereof. The particle may include, in addition to the therapeuticagent(s), any of those materials routinely used in the art of pharmacyand medicine, including, but not limited to, erodible, nonerodible,biodegradable, or nonbiodegradable material or combinations thereof. Theparticles may be microcapsules that contain the kinase inhibitingcomposition in a solution or in a semi-solid state. The particles may beof virtually any shape.

Both non-biodegradable and biodegradable polymeric materials may be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired.Bioadhesive polymers of particular interest include bioerodiblehydrogels as described by Sawhney et al in Macromolecules (1993) 26,581-587, the teachings of which are incorporated herein. These includepolyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems.In order to prolong the effect of a drug, it often is desirable to slowthe absorption of the drug from subcutaneous, intrathecal, orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. The term “controlled release” is intended to refer toany drug-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. This refers to immediate aswell as non-immediate release formulations, with non-immediate releaseformulations including, but not limited to, sustained release anddelayed release formulations. The term “sustained release” (alsoreferred to as “extended release”) is used herein in its conventionalsense to refer to a drug formulation that provides for gradual releaseof a drug over an extended period of time, and that preferably, althoughnot necessarily, results in substantially constant blood levels of adrug over an extended time period. Alternatively, delayed absorption ofa parenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. The term “delayed release” isused herein in its conventional sense to refer to a drug formulation inwhich there is a time delay between administration of the formulationand the release of the drug there from. “Delayed release” may or may notinvolve gradual release of drug over an extended period of time, andthus may or may not be “sustained release.”

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. The term “long-term”release, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 7 days, and preferably about 30 to about 60 days. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

In another aspect, the present invention further provides a biomedicaldevice comprising at least one isolated kinase inhibiting peptidecomprising a sequence according to Formula IV, wherein the one or moreisolated kinase inhibiting peptides are disposed on or in the device. Insome such embodiments, the at least one kinase inhibiting peptide is atleast one peptide having an amino acid sequence selected from the groupconsisting of HRRIKAWLKKILALARQLGVAA [SEQ ID NO: 166];WLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113]; and WLRRIKAWLRRALARQLGVA [SEQID NO: 177]. In some such embodiments, the biomedical device comprisesat least one isolated kinase inhibiting peptides comprising a sequenceaccording to Formula V, wherein the one or more isolated kinaseinhibiting peptides are disposed on or in the device. In some suchembodiments, the biomedical device comprises at least one isolatedkinase inhibiting peptide comprising a sequence according to Formula VI,wherein the one or more isolated kinase inhibiting peptides are disposedon or in the device. In some such embodiments, the kinase inhibitingpeptide is a peptide having an amino acid sequence selected from thegroup consisting of KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173];FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]; and WLRRIKAWLRRIKALNRQLGVAA [SEQID NO: 142].

General methods in molecular genetics and genetic engineering useful inthe present invention are described in the current editions of MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P.Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2^(nd)Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transferand Expression Protocols, pp. 109-128, ed. E. J. Murray, The HumanaPress Inc., Clifton, N.J.). Reagents, cloning vectors, and kits forgenetic manipulation are available from commercial vendors such asBioRad, Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co.

Where a value of ranges is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and” and “the” include plural references unless thecontext clearly dictates otherwise. All technical and scientific termsused herein have the same meaning

Publications disclosed herein are provided solely for their disclosureprior to the filing date of the present invention. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Comparison of the Effectiveness of a Peptide Inhibitor ofMAPKAP Kinase II (MK2) Alone or Covalently Bound to the WLRRIKAWLRRIKA[SEQ ID NO: 134] Transduction Domain

Using the Omnia™ Lysate Assay for MAPKAP-K2 kit (Invitrogen, Carlsbad,Calif.), the reaction velocity for MK2 was determined in the presenceand absence of each of the peptides listed in Table 2. Briefly,inhibitor peptide concentrations at 12.5 μmol, 25 μmol, 50 μmol and 100μmol were evaluated. The kit contains a proprietary reaction buffer towhich the following are added (final concentrations are given): 1 mMATP, 0.2 mM DTT, 10 μM MAPKAP-K2 Sox-modified peptide substrate, 5 ngMK2, and the peptide inhibitor of interest (final volume of 50 μL). Thereactions were performed in the wells of a low protein-binding 96-wellplate provided with the kit, and fluorescence readings at 485 nm weretaken every 30 seconds for 20 minutes in a Molecular Devices M5Spectrophotometer. A peptide having the sequence KALNRQLGVAA [SEQ ID NO:124] was used as a baseline because it is a known inhibitor based on thework of Hayess and Benndorf (Katrin Hayess and Rainer Benndorf, 1997,“Effect of protein kinase inhibitors on activity of mammalian smallheat-shock protein (HSP25) kinase”, Biochemical Pharmacology, 53(9):1239-1247.

The reaction velocities for a MK2 inhibitor concentration of 100 μM areshown in Table 3; relative reaction velocities between peptides weresimilar at lower and higher concentrations (data not shown). The resultsof this study show that several of the kinase inhibiting peptidesaccording to the present invention were able to inhibit MK2 activity.The results further provide information as to the amino acids that aremost crucial for the function of the MK2 inhibitor. Note that thecovalent attachment of the MK2 inhibitor peptide to the WLRRIKAWLRRIKA[SEQ ID NO: 134] transduction domain substantially increases the MK2inhibitor function.

TABLE 2 MK2 Inhibitor Sequence Evaluation  of Each Amino Acid[SEQ ID NO:] Controls KALNRQLGVA* [SEQ ID NO: 114] KKKALNRQLGVAA^(#)[SEQ ID NO: 115] (WLRRIKA)₂LNRQLGVAA [SEQ ID NO: 178] Alanine ScanKALNRQLGVAA [SEQ ID NO: 116] KA A NRQLGVAA [SEQ ID NO: 117] KAL ARQLGVAA [SEQ ID NO: 118] KALN A QLGVAA [SEQ ID NO: 119] KALNR A LGVAA[SEQ ID NO: 120] KALNRQ A GVAA [SEQ ID NO: 121] KALNRQL A VAA[SEQ ID NO: 122] KALNRQLG A AA [SEQ ID NO: 123] d-Amino Acid Scan K dALNRQLGVAA [SEQ ID NO: 179] K Ad LNRQLGVAA [SEQ ID NO: 180] KAL dNRQLGVAA [SEQ ID NO: 181] KALN dR QLGVAA [SEQ ID NO: 182] Controls KALNRdQ LGVAA [SEQ ID NO: 183] KALNRQ dL GdVAA [SEQ ID NO: 184] KALNRQLG dVAA [SEQ ID NO: 185] * = Control to determine the requirement of thefinal A; ^(#) = Control to determine the importance of initial Ks

TABLE 3 Reaction Velocities for MK2 Inhibitor  Variants (n = 3)% of KALNRQLGVAA  [SEQ ID NO: 124] Reaction Velocity  at an InhibitorConcentration of  100 μM  MK2 Inhibitor Variant (+/−Peptide Peptide Sequence Sequence SEM*) WLKKIKAWLKKIKALNRQLGVVA  −32% (+/−6%)[SEQ ID NO: 159] KALNRQLGVAA [SEQ ID NO: 124] 100% (+/−3%)KALNRQLGVA [SEQ ID NO: 125] 100% (+/−3%) KAANRQLGVAA [SEQ ID NO: 126]152% (+/−3%) KALARQLGVAA [SEQ ID NO: 127]  39% (+/−1%)KALNAQLGVAA [SEQ ID NO: 128] 358% (+/−8%) KALNRALGVAA [SEQ ID NO: 129]358% (+/−15%) KALNRQAGVAA [SEQ ID NO: 130] 118% (+/−4%)KALNRQLAVAA [SEQ ID NO: 131]  72% (+/−3%) KALNRQLGAAA [SEQ ID NO: 132]373% (+/−13%) KAdLNRQLGVAA [SEQ ID NO: 146% (+/−4%) KALdNRQLGVAA 95% (+/−6%) KALNdRQLGVAA 306% (+/−4%) KALNRdQLGVAA 276% (+/−3%)KALNRQdLGVAA 357% (+/−10%) KALNRQLGdVAA 260% (+/−14%) KKKALNRQLGVAA  91% (+/−4%) [SEQ ID NO: 133] *SEM = Standard Error of the Mean forthree values

Example 2 Transduction Domain Reaction Velocities

The MK2 inhibition activity of WLRRIKAWLRRIKA [SEQ ID NO: 134]transduction domain was compared with the MK2 inhibition activity ofother known transduction domains—YARAAARQARA [SEQ ID NO: 135] andYGRKKKRRQRRR [SEQ ID NO: 136]. The transduction domains were tested forMK2 activity using the same assay conditions as Example 1. The resultsare shown in Table 4.

TABLE 4 Reaction Velocities for Different Transduction Domains (n = 3)% of KKKALNRQLGVAA [SEQ ID NO: 115] Reaction Velocity [SEQat an Inhibitor Peptide ID Concentration of Sequence NO:]100 μM (+/−SEM*) WLRRIKA 137 394% (+/−5%) WLRRIKAWLRRIKA 134 19% (+/−2%) YARAAARQARA 135 274% (+/−9%) YGRKKRRQRRR 136 158% (+/−11%)*SEM = Standard Error of the Mean for three values

These results indicate substantial differences in the effect of severaltransduction domains on inhibiting MK2 and that transduction domains areuseful to inhibit the activity of kinases, such as MK2. The WLRRIKA [SEQID NO: 137] monomer had the same level of inhibition as the no inhibitorcontrol (data not shown); however, the WLRRIKAWLRRIKA [SEQ ID NO: 134]transduction domain dimer inhibited MK2 significantly more thanKKKALNRQLGVAA [SEQ ID NO: 115]. YARAAARQARA [SEQ ID NO: 135] andYGRKKRRQRRR [SEQ ID NO: 136] also inhibit MK2 but not nearly as much asWLRRIKAWLRRIKA [SEQ ID NO: 134].

Example 3 MK2 Inhibitor Peptides with Transduction Domains

A set of MK2 inhibitor peptides having transduction domains wassynthesized (see Table 5). Variants included peptides with theWLRRIKAWLRRIKA [SEQ ID NO: 134] transduction domain and alaninesubstituted for asparagine, alanine substituted for glycine, or bothalanine substitutions in the therapeutic domain. The same peptides withthe YARAAARQARA [SEQ ID NO: 135] transduction domain also were prepared.The reaction velocities of these variants were compared to the reactionvelocity of a blank with no inhibitor added. Results are shown in Table6. The data in Table 6 shows a synergy between the transduction domainand the therapeutic domain in inhibiting MK2. Since the WLRRIKAWLRRIKA[SEQ ID NO: 134] transduction domain is a much stronger inhibitor of MK2than the YARAAARQARA [SEQ ID NO: 135] transduction domain, peptides witha WLRRIKAWLRRIKA [SEQ ID NO: 134] transduction domain are much strongerinhibitors of MK2 at a given concentration than are peptides with theYARAAARQARA [SEQ ID NO: 135] transduction domain.

TABLE 5 Proposed Optimized Therapeutic Domains with Two Different Transduction Domains Peptides with Peptides withWLRRIKAWLRRIKA YARAAARQARA [SEQ ID NO: 134] SEQ [SEQ ID NO: 135] SEQ Transduction Domain ID NO: Transduction Domain ID NO:WLRRIKAWLRRIKALARQLAVA 138 YARAAARQARAKALARQLAVA 143WLRRIKAWLRRIKALARQLGVA 139 YARAAARQARAKALARQLGVA 144WLRRIKAWLRRIKALARQLAVA 140 YARAAARQARAKALNRQLAVA 145WLRRIKAWLRRIKALNRQLGVA 141 YARAAARQARAKALNRQLGVA 146WLRRIKAWLRRIKALNRQLGVAA 142 YARAAARQARAKALNRQLGVAA 147

TABLE 6 Reaction Velocities for Selected Optimized Peptides (n = 3)% of Blank Reaction Velocity at an Inhibitor Concentration SEQof Peptide ID Sequence 6.25 Peptide Sequence NO pN (+/− SEM*)WLRRIKAWLRRIKALARQLAVA 138  3.3% (+/−0.4%) WLRRIKAWLRRIKALARQLGVA 139 3.9% (+/−0.8%) WLRRIKAWLRRIKALNRQLAVA 140 −2.1% (+/−0.5%)WLRRIKAWLRRIKKKALARQLAVA 148  1.0% (+/−0.4%) YARAAARQARAKALARQLGVA 14328.9% (+/−0.8%) YARAAARQARAKKKALARQLAVA 112 22.4% (+/−0.5%)No Inhibitor Added —  100% (+/−2%) *SEM = Standard Error of the Mean orthree values

Example 4 Kinase Profiler Assays

KIP peptides WLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142],KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173], and FAKLAARLYRKALARQLGVAA [SEQID NO: 163] were utilized to profile a variety of different kinases. Thekinase profiler assay services provided by Upstate, a part of MilliporeCorporation (Billerica, Mass.), were utilized to conduct the kinaseprofiles. The kinase profiler assays, which are radiometric, are basedon measuring the amount of radioactive transfer from an ATP (γ-³³P-ATP)to a substrate peptide using known concentrations of kinase enzyme,inhibitor, ATP, and buffer at defined time points and temperatures.Details on the reaction conditions for each kinase are available athttp://www.millipore.com/drugdiscovery/dd3/KinaseProfiler. For example,the inhibition of ROCK-1(h) was tested using a buffer containing 20 mMMOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1% β-mercaptoethanol, 1mg/mL BSA. ROCK-1(h) (5-10 mU) was incubated with 8 mM MOPS pH 7.0, 0.2mM EDTA, 30 μM of the peptide KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 10 mM Mgacetate and γ-³³P-ATP (specific activity approximately 500 cpm/pmol,concentration as required) in a final reaction volume of 25 μl. Thereaction was initiated by the addition of the MgATP mixture in theabsence and presence of inhibitor compounds. After incubation for 40minutes at room temperature, the reaction was stopped by the addition of5 μl of a 3% phosphoric acid solution. 10 μl of the reaction mixturethen was spotted onto a P30 filtermat. The filtermat was washed threetimes for 5 minutes in 75 mM phosphoric acid, once in methanol, driedand counted by scintillation counting. Where the kinase is uninhibited,the kinase phosphorylates a positively charged substrate withradioactive ATP, which then binds to a negatively charged filtermembrane. Scintillation counts (radioactivity) directly correlate withkinase activity. The assay was run at an ATP concentration within 15micromolar of the K_(m) for each individual kinase. All profile dataless than 100 indicate kinase inhibition while data greater than 100indicate kinase stimulation.

The results of the kinase profiler assay are shown in Table 7 forWLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142]; in Table 8 forKAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173]; and in Table 9 forFAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]. The results show that KIPpeptides are useful in regulating kinase function. More specifically,these data show that WLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142],KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173], and FAKLAARLYRKALARQLGVAA [SEQID NO: 163] peptides inhibit several kinases, many of which are known tobe important mediators of hyperplasia and cancer.

TABLE 7 Results of the kinase profiler assay using test peptide WLRRIKAWLRRIKALNRQLGVAA [SEQ ID NO: 142]. Profile-1 Kinase@ = 30 μm Abl(h)  24 AMPK (r)  36 Aurora-A(h)  46 BTK(h)   5 CaMKI (h)  9 CDKI/cyclinB(h)  17 CHK1(h)  31 CKIB (h)  52 CK2 (h) 114 cKit(h)  23DYRK2 (h)  93 EGFR(h)  10 EphA2 (h)  38 FGFR1(h)  27 Flt3 (h)  38 GSK3β(h) 129 IGF-IR(h) 227 IRAK4 (h)  12 JAK3 (h)  85 KDR(t1)  27 Lck (h) 130L1MK1 (h)  89 MAPKI (h) 121 MEKI (h)  14 Met (h)  30 MLCK(h)   4PDGFRβ(h)  42 PhKγ2 (h)  15 Pim-1 (h)   5 PKA(h)  80 PKB β (h)  18 PKC β(h)   8 PKCδ (h)  11 PKG1α(h)  16 PKG1β(h)  15 Ret (h) 117 ROCK-1(h)   0Rsk2 (h)  14 SAPK2 a (h)  61 Src(1-530)(h)   6 Syk(h)  19 Tie2 (h)  17TrkA(h)   6

TABLE 8 Results of the kinase profiler assay using test peptide KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173] Profile-1 Kinase @= 100 μm Abl(h)  55 AMPK (r) 118 ASK1(h)  48 Aurora-A(h)  60 BTK(h)  16CaMKI (h)   0 CDKI/cyclinB(h)  55 CHK1(h)  68 CKIB (h)  97 CK2 (h)  79cKit(h)  31 DYRK2 (h) −10 EGFR(h)  16 EphA2 (h)  22 FGFR1(h)  35Flt3 (h)  20 GSK3β (h) 184 IGF-IR(h)  76 IRAK4 (h)  16 JAK3 (h)  91JNK1α1(h)  92 KDR(t1)  56 Lck (h) 847 L1MK1 (h)  93 MAPKI (h) 108MAPKAP-   8 K2(h) MAPKAP-  17 K3 (h) MEKI (h)  66 Met (h)  22 MKK4(m)114 MKK6(h)  48 MLCK(h)   2 MSK1(h)  13 MSK2(h)  30 PDGFRβ(h)  66PhKγ2 (h)  27 Pim-1 (h)   1 PKA(h) 103 PKB β (h)  28 PKC β 1 (h)  23PKCδ (h)  24 PKG1α(h)  25 PKG1β (h)  24 PRAK 148 Ret (h) 117 ROCK-1(h) 29 Rsk2 (h)   6 SAPK2 a (h)  59 Src(1-530)(h)   3 Syk(h)   4 Tie2 (h)  8 TrkA(h)  16

TABLE 9 Results of the kinase profiler assay usingtest peptide FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163]. Profile-1 Kinase @= 100 μm Abl(h)  41 AMPK(r) 101 ASK1(h)  53 Aurora-A(h)  65 BTK(h)  19CaMKI(h)   0 CDKI/cyclinB(h)  36 CHK1(h)  54 CKIB(h)  99 CK2(h)  80cKit(h)  42 DYRK2(h) −11 EGFR(h)  18 EphA2 (h)  32 FGFR1(h)  22 Flt3 (h) 14 GSK3β (h) 188 IGF-IR(h)  69 IRAK4 (h)  13 JAK3 (h) 102 JNK1α1(h)  91KDR(t)  78 Lck (h) 493 L1MK1 (h)  92 MAPKI (h) 104 MAPKAP-   5 K2(h)MAPKAP-  10 K3(h) MEKI (h)  68 Met (h)  17 MKK4(m)  90 MKK6(h)  42MLCK(h)   1 MSK1(h)  11 MSK2(h)  24 PDGFRβ(h)  92 PhKγ2 (h)  20Pim-1 (h)   1 PKA(h)  76 PKB β (h)  16 PKC β1 (h)  73 PKC δ (h)  40PKG1α(h)  12 PKG1β (h)  15 PRAK 131 Ret (h)  89 ROCK-1(h)  25 Rsk2 (h) −1 SAPK2a (h)  30 Src(1-530)(h)   5 Syk(h)  38 Tie2 (h)   0 TrkA(h)  17

Example 5 Toxicity Testing of KIP Peptides on Multiple Cancer Cell Lines

The effect of six concentrations of four KIP peptides on seven cancercell lines was evaluated. The KIP peptides used in these experiments arelisted in Table 10.

TABLE 10 KIP Peptides tested on cancer cell lines SEQ Peptide ID NumberPeptide Primary Structure NO: 1 HRRIKAWLKKIKALARQLGVAA 166 2WLRRIKAHRRIKALARQLGVAA 167 3 WLRRIKAWLRR 168 4 WLRRIKAWLRRALNRQLGVAA 169

The seven cell lines were: estrogen dependent MCF-7 breast cancer cells,non estrogen dependent MDA 231 breast cancer cells, SF 539 centralnervous system cancer cells, HT29 colon cancer cells, Paca 2 pancreaticcancer cells, PC3 prostate cancer cells, and A549 lung cancer cells. Foreach cell line, exponentially growing cells were trypsinized and seededinto individual wells of a 96-well plate. Cells were maintained in ahumidified, 5% CO₂ incubator at 37° C. for 24 hours. The next day, freshmedia was added along with 1 μl of a stock solution of peptide to givefinal peptide concentrations of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and100 μM (n=4). Doxorubicin was tested as a positive control. Afterincubating the cells for 72 hours, to each well was added 20 μl of 0.5%MTT {3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide}solution. The plates then were incubated for an additional 4 hours, atwhich time the absorbance at 570 nm was measured for each well using amicroplate reader. The aborbance was plotted as function of peptideconcentration, and IC50 values were calculated for each peptide for eachcell line. The results from this experiment are presented in FIGS. 1-7.Calculated IC50 values are presented in Table 11. For all cell lines,the IC50 values for each of the four peptides was below 50 μM. Theseresults indicate that KIP peptides are antiproliferative/cytotoxic toseveral different cancer cell lines in a dose dependent manner.

TABLE 11 IC50 for KIP peptides tested on cancer cell lines Cell LineIC50 (μM) Peptide MCF-7 MDA 231 SF 539 HT29 Paca 2 A549 PC3 Peptide 11.6 9.3 4.3 6.7 9.0 8.7 6.0 Peptide 2 7.8 19.9 26.0 26.4 19.5 31.9 28.9Peptide 3 13.5 17.4 22.0 20.7 42.5 34.3 35.5 Peptide 4 4.4 12.0 10.011.8 11.2 12.5 23.7

Example 6 Evaluation of Apoptosis in MCF-7 Breast Cancer Cells Using aKIP Peptide

MCF-7 breast cancer cells were seeded in individual wells of a 96-wellplate and were maintained for 24 hours in a humidified, 5% CO2 incubatorat 37° C. The next day, fresh media was added along with 1 μl of a stocksolution of KIP peptide HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166] to givea final concentration of 3 μM or 10 μM (n=4). After incubating the cellsfor 24 hours, cells were treated with Hoescht dye for nuclear staining,propidium iodide to stain for DNA from necrotic cells, and Annexin V tovisualize apoptotic cells.

As seen in FIG. 8, MCF-7 breast cancer cells have a high degree ofAnnexin V staining relative to propidium iodide staining for bothconcentrations of KIP peptide. These results indicate that KIP peptidescan induce apoptosis in a cancer cell line.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-134. (canceled)
 135. A method for inhibiting a kinase activity of akinase enzyme, the method comprising the step of providing a kinaseinhibiting composition, wherein the kinase inhibiting compositioncomprises an inhibitory amount of a kinase inhibiting peptide, whereinthe kinase inhibiting peptide comprises an amino acid sequence accordingto Formula VI: Z1-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-Z2 [Formula VI] [SEQ IDNO: 226], wherein each of Z1 and Z2 is absent or is a transductiondomain; X1 is KA; X2 is L; X3 is A; X4 is R; X5 is selected from thegroup consisting of Q and N; X6 is L; X7 is G; X8 is V; X9 is A; X10 isA, and wherein the kinase enzyme contacts the kinase inhibitingcomposition, wherein the activity of the kinase enzyme is inhibited, andwherein the kinase enzyme is selected from the group consisting of: Ab1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI,CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin,CK1y1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR,DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit,Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7, GSK, CSK3, Hck,HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK, Insulin R, IRAK,IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR, Lck, LIMK, LIMK1,LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK, Met, Mer, MINK,MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα,PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1,PKCδ, PKD2, PKR, PKA, PKBβ, PKCβ1, PKG1, PKG1α, PKG1β, PLK, PRAK, PTK5,Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAPKinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src, Syk, TAK1,TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3, Ulk2,VRK2, WEE, Yes, ZAP-70 and ZIPK.
 136. The method according to claim 135,wherein the amino acid sequence of the kinase inhibiting peptide isWLRRIKAWLRRIKALARQLGVAA [SEQ ID NO: 113].
 137. The method according toclaim 135, wherein the amino acid sequence of the kinase inhibitingpeptide is KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173].
 138. The methodaccording to claim 135, wherein the amino acid sequence of the kinaseinhibiting peptide is FAKLAARLYRKALARQLGVAA [SEQ ID NO: 163].
 139. Themethod according to claim 135, wherein the kinase inhibiting peptide isHRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166].
 140. The method according toclaim 135, wherein the kinase inhibiting peptide isYARAAARQARAKALARQLGVAA [SEQ ID NO: 106].
 141. A method for inhibitinghyperplasia in a cell population, the method comprising the step ofproviding a therapeutically effective amount of a kinase inhibitingcomposition to a subject in need thereof, wherein the kinase inhibitingcomposition comprises an inhibitory amount of a kinase inhibitingpeptide, wherein the kinase inhibiting peptide inhibits a kinaseactivity of a kinase enzyme, wherein the kinase enzyme is selected fromthe group consisting of: Ab 1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl,Blk, Bmx, Brk, BTK, CaMKI, CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, CaseinKinase, Cdk, CDK9/cyclin, CK1y1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK,CDK1/cyclinB, CHK1, CHK2 mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott,Csk, DAPK1, DCAMKL2, DDR, DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps,FGFR, FGFR1, Fgr, Fit, Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7,GSK, CSK3, Hck, HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK,Insulin R, IRAK, IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR,Lck, LIMK, LIMK1, LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK,Met, Mer, MINK, MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3,NEK9, PDGFR, PDGFRα, PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2,Pim-3, PKC, PKCβ1, PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI, PKG1, PKG1α,PKG1β, PLK, PRAK, PTK5, Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron,Ros, Rse, Rsk4, Rsk/MAPKAP Kinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src,Src(1-530), Src, Syk, TAK1, TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2,TrkA, Txk, ULK3, Ulk2, VRK2, WEE, Yes, ZAP-70 and ZIPK, wherein thekinase inhibiting peptide is YARAAARQARAKALARQLGVAA [SEQ ID NO: 106],wherein at least one hyperplastic cell contacts the kinase inhibitingcomposition, and wherein the hyperplasia of the at least onehyperplastic cell is inhibited.
 142. A method for inhibiting growth of aneoplasm, the method comprising the step of providing a therapeuticallyeffective amount of a kinase inhibiting composition to a subject in needthereof, wherein the kinase inhibiting composition comprises aninhibitory amount of a kinase inhibiting peptide, wherein the kinaseinhibiting peptide inhibits a kinase activity of a kinase enzyme,wherein the kinase enzyme is selected from the group consisting of: Ab1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI,CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin,CK1y1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR,DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit,Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7, GSK, CSK3, Hck,HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK, Insulin R, IRAK,IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR, Lck, LIMK, LIMK1,LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK, Met, Mer, MINK,MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα,PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1,PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI, PKG1, PKG1α, PKG1β, PLK, PRAK, PTK5,Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAPKinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src, Syk, TAK1,TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3, Ulk2,VRK2, WEE, Yes, ZAP-70 and ZIPK, wherein the amino acid sequence of thekinase inhibiting peptide is selected from the group consisting ofFAKLAARLYRKALARQLGVAA [SEQ ID NO: 163] and HRRIKAWLKKIKALARQLGVAA [SEQID NO: 166], wherein the kinase inhibiting composition contacts theneoplasm, and wherein the growth of the neoplasm is inhibited.
 143. Themethod according to claim 142, wherein the neoplasm is a carcinoma. 144.The method according to claim 142, wherein the neoplasm is selected fromthe group consisting of a breast cancer, a central nervous systemcancer, a colon cancer, a pancreatic cancer, a prostate cancer, and alung cancer.
 145. The method according to claim 142, wherein the kinaseinhibiting peptide is HRRIKAWLKKIKALARQLGVAA [SEQ ID NO: 166].
 146. Themethod according to claim 142, wherein the kinase inhibiting peptide isFAKLAARLYRKALARQLGVAA [SEQ ID NO: 163].
 147. A method for inducingprogrammed cell death in a cell population, the method comprising thestep of providing a kinase inhibiting composition, wherein the kinaseinhibiting composition comprises an inhibitory amount of a kinaseinhibiting peptide, wherein the kinase inhibiting peptide inhibits akinase activity of a kinase enzyme, wherein the kinase enzyme isselected from the group consisting of: Ab 1, Akt/PKB, AMPK, Arg, Ask,Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI, CaMKIδ, CaMKIIβ, CaMKIIγ,CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin, CK1y1, CK1y2, CK1y3, Ck1δ,CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2 mutants, CK1δ, CK2, c-Kit,CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR, DYRK2, EGFR, Ephs, EphA2,FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit, Flt3, Flt4, Fms/CSF-1 R, Fyn,GRK5, GRK6, GRK7, GSK, CSK3, Hck, HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1,ICF IR, IKK, Insulin R, IRAK, IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3,JNK/SAPK, KDR, Lck, LIMK, LIMK1, LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase,MEK, MEK1, MELK, Met, Mer, MINK, MKK, MLCK, MLK1, MRCKa, MSK1, MST,MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα, PDGFRβ, PDK, PhKγ2, PI 3-Kinase,PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1, PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI,PKG1, PKG1α, PKG1β, PLK, PRAK, PTK5, Pyk, Raf, Rct, RIPK2, ROK/ROCK,ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAP Kinase, S6 Kinase, Rsk2, SAPK2a,SGK, c-Src, Src(1-530), Src, Syk, TAK1, TAO1, TAO2, TBK, Tie2/TEK, TLK2,Trk, TSSK2, TrkA, Txk, ULK3, Ulk2, VRK2, WEE, Yes, ZAP-70 and ZIPK,wherein the kinase inhibiting peptide is HRRIKAWLKKIKALARQLGVAA [SEQ IDNO: 166], wherein the kinase inhibiting composition contacts at leastone cell in the cell population, and wherein programmed cell death ofthe at least one cell is induced.
 148. The method according to claim147, wherein the cell is a prokaryotic cell.
 149. The method accordingto claim 147, wherein the cell is a eukaryotic cell.
 150. The methodaccording to claim 147, wherein the programmed cell death occurs byapoptosis.
 151. A method for inhibiting growth of a neoplasm, the methodcomprising the step of providing a therapeutically effective amount of akinase inhibiting composition to a subject in need thereof, wherein theneoplasm is selected from the group consisting of a melanoma, a prostatecancer, and a lung cancer, wherein the kinase inhibiting compositioncomprises an inhibitory amount of a kinase inhibiting peptide, whereinthe kinase inhibiting peptide inhibits a kinase activity of a kinaseenzyme, wherein the kinase enzyme is selected from the group consistingof: Ab 1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl, Blk, Bmx, Brk, BTK,CaMKI, CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, Casein Kinase, Cdk,CDK9/cyclin, CK1y1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK, CDK1/cyclinB,CHK1, CHK2 mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott, Csk, DAPK1,DCAMKL2, DDR, DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps, FGFR, FGFR1,Fgr, Fit, Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7, GSK, CSK3,Hck, HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK, Insulin R, IRAK,IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR, Lck, LIMK, LIMK1,LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK, Met, Mer, MINK,MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα,PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1,PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI, PKG1, PKG1α, PKG1β, PLK, PRAK, PTK5,Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAPKinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src, Syk, TAK1,TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3, Ulk2,VRK2, WEE, Yes, ZAP-70 and ZIPK, wherein the amino acid sequence of thekinase inhibiting peptide is KAFAKLAARLYRKALARQLGVAA [SEQ ID NO: 173],wherein the kinase inhibiting composition contacts the neoplasm, andwherein the growth of the neoplasm is inhibited.
 152. A method forinhibiting growth of a neoplasm, the method comprising the step ofproviding a therapeutically effective amount of a kinase inhibitingcomposition to a subject in need thereof, wherein the neoplasm is abreast cancer, wherein the kinase inhibiting composition comprises aninhibitory amount of a kinase inhibiting peptide, wherein the kinaseinhibiting peptide inhibits a kinase activity of a kinase enzyme,wherein the kinase enzyme is selected from the group consisting of: Ab1, Akt/PKB, AMPK, Arg, Ask, Aurora-A, Axl, Blk, Bmx, Brk, BTK, CaMKI,CaMKIδ, CaMKIIβ, CaMKIIγ, CaMKI1β, Casein Kinase, Cdk, CDK9/cyclin,CK1y1, CK1y2, CK1y3, Ck1δ, CK2α, CK2, CHK, CDK1/cyclinB, CHK1, CHK2mutants, CK1δ, CK2, c-Kit, CLK2, CLK3, Cott, Csk, DAPK1, DCAMKL2, DDR,DYRK2, EGFR, Ephs, EphA2, FAK, Fer, Fes/Fps, FGFR, FGFR1, Fgr, Fit,Flt3, Flt4, Fms/CSF-1 R, Fyn, GRK5, GRK6, GRK7, GSK, CSK3, Hck,HER/ErbB, HIPK1, HIPK2, HIPK3, IGF-1, ICF IR, IKK, Insulin R, IRAK,IRAK1, IRAK4, JAK, JAK1, JAK2, JAK3, JNK/SAPK, KDR, Lck, LIMK, LIMK1,LOK, Lyn, MAPK, MAPK1, MAPKAP Kinase, MEK, MEK1, MELK, Met, Mer, MINK,MKK, MLCK, MLK1, MRCKa, MSK1, MST, MST3, NEK, NEK3, NEK9, PDGFR, PDGFRα,PDGFRβ, PDK, PhKγ2, PI 3-Kinase, PIM, Pim-1, Pim-2, Pim-3, PKC, PKCβ1,PKCδ, PKD2, PKR, PKA, PKBβ, PKCβI, PKG1, PKG1α, PKG1β, PLK, PRAK, PTK5,Pyk, Raf, Rct, RIPK2, ROK/ROCK, ROCK-I, Ron, Ros, Rse, Rsk4, Rsk/MAPKAPKinase, S6 Kinase, Rsk2, SAPK2a, SGK, c-Src, Src(1-530), Src, Syk, TAK1,TAO1, TAO2, TBK, Tie2/TEK, TLK2, Trk, TSSK2, TrkA, Txk, ULK3, Ulk2,VRK2, WEE, Yes, ZAP-70 and ZIPK, wherein the amino acid sequence of thekinase inhibiting peptide is YARAAARQARAKALARQLGVAA [SEQ ID NO: 106],wherein the kinase inhibiting composition contacts the neoplasm, andwherein the growth of the neoplasm is inhibited.